Methods of making and using extracellular domain-based chimeric proteins

ABSTRACT

The present invention relates, inter alia, to compositions and methods, including chimeric proteins and combination therapies that find use in the treatment of disease, such as cancer and/or an inflammatory disease.

PRIORITY

This application is a continuation of U.S. application Ser. No.16/484,852, filed Aug. 9, 2019, now U.S. Pat. No. 11,332,509. U.S.application Ser. No. 16/484,852 is a 371 national stage application ofPCT/US18/20040 filed Feb. 27, 2018, which claims the benefit of, andpriority to, U.S. Provisional Application No. 62/464,002, filed Feb. 27,2017, the contents of each of which are hereby incorporated by referencein its entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

This application contains a sequence listing. It has been submittedelectronically via EFS-Web as an ASCII text file entitled“SHK-001US2C1_SequenceListing_ST25”. The sequence listing is 149,185bytes in size, and was created on or about Apr. 11, 2022. The sequencelisting is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present invention relates to, inter alia, compositions and methods,including chimeric proteins that find use in the treatment of disease,such as immunotherapies for cancer and autoimmunity.

BACKGROUND

Exceptional treatment for cancer have been evasive despite years ofefforts. This is so, in part, because many cancers have developedmechanisms to avoid the immune system. Thus, there remains a need todevelop therapeutics and treatment regimens that adequate engagemultiple arms of the immune system to generate an anti-cancer immuneresponse.

SUMMARY

Accordingly, in various aspects, the present invention provides forcompositions and methods that are useful for cancer immunotherapy. Forinstance, the present invention, in part, relates to specific chimericproteins that reverse or suppresses immune inhibitory signals whileproviding immune activating or co-stimulatory signals. Importantly,inter alia, the present invention provides for improved chimericproteins that can maintain a stable and producible multimeric statebased on, without wishing to be bound by theory, stabilization in alinker region including one or more disulfide bonds. Accordingly, thepresent compositions and methods overcome various deficiencies inproducing bi-specific agents.

In some aspects, the chimeric protein is of a general structure of: Nterminus-(a)-(b)-(c)-C terminus, where (a) is a first domain comprisingan extracellular domain of a Type I transmembrane protein, (b) is alinker comprising at least one cysteine residue capable of forming adisulfide bond (including without limitation, hinge-CH2-CH3 Fc domain isderived from human IgG4), and (c) is a second domain comprising anextracellular domain of Type II transmembrane protein, where the linkerconnects the first domain and the second domain and optionally comprisesone or more joining linkers as described herein.

Further, in various aspects, the present invention relates to methods oftreating cancer by using a combination of the present chimeric proteins.For example, the present methods allow for regimens that modulatespecific arms of the immune system, such as innate and adaptive immuneresponses, optionally in order.

In some aspects, there is provided a method of treating cancer,comprising administering to a subject in need thereof: (i) a firstchimeric protein comprising a general structure of Nterminus-(a)-(b)-(c)-C terminus, where: (a) is a first domain comprisingan extracellular domain of a Type I transmembrane protein, (b) is alinker comprising at least one cysteine residue capable of forming adisulfide bond, and (c) is a second domain comprising an extracellulardomain of a Type II transmembrane protein, and the first chimericprotein modulates the innate immune system; and (ii) a second chimericprotein comprising a general structure of N terminus-(a)-(b)-(c)-Cterminus, where (a) is a first domain comprising an extracellular domainof a Type I transmembrane protein, (b) is a linker comprising at leastone cysteine residue capable of forming a disulfide bond, and (c) is asecond domain comprising an extracellular domain of Type IItransmembrane protein, and the second chimeric protein modulates theadaptive immune system.

In some aspects, there is provided a method of treating cancer,comprising administering to a subject in need thereof: a second chimericprotein comprising a general structure of N terminus-(a)-(b)-(c)-Cterminus, where (a) is a first domain comprising an extracellular domainof a Type I transmembrane protein, (b) is a linker comprising at leastone cysteine residue capable of forming a disulfide bond, and (c) is asecond domain comprising an extracellular domain of Type IItransmembrane protein, and the second chimeric protein modulates theadaptive immune system, where the subject is undergoing or has undergonetreatment with a first chimeric protein comprising a general structureof N terminus-(a)-(b)-(c)-C terminus, where (a) is a first domaincomprising an extracellular domain of a Type I transmembrane protein,(b) is a linker comprising at least one cysteine residue capable offorming a disulfide bond, and (c) is a second domain comprising anextracellular domain of Type II transmembrane protein, and the firstchimeric protein modulates the innate immune system.

In embodiments, first chimeric protein is administered before the secondchimeric protein.

In embodiments, the first chimeric protein is administered after thesecond chimeric protein.

In embodiments, the first chimeric protein comprises at least one of:TIGIT, CSF1R, CD172a (SIRP1α), VSIG8, TIM3, 41BBL, CD40L, SIGLEC7,SIGLEC9, and LIGHT.

In embodiments, the second chimeric protein comprises at least one of:PD-1, TIM3, VSIG8, CD172a (SIRP1α), OX40L, GITRL, TL1A, and IL-2

In embodiments, the first chimeric protein and the second chimericprotein are independently selected from TIM3-Fc-OX40L, CD172a(SIRP1α)-Fc-CD40L, and CSF1R-Fc-CD40L.

In embodiments, TIM3-Fc-OX40L is administered before CD172a(SIRP1α)-Fc-CD40L. In embodiments, TIM3-Fc-OX40L is administered beforeCSF1R-Fc-CD40L. In embodiments, CD172a (SIRP1α)-Fc-CD40L is administeredbefore TIM3-Fc-OX40L. In embodiments, CSF1R-Fc-CD40L is administeredbefore TIM3-Fc-OX40L.

Any aspect or embodiment described herein can be combined with any otheraspect or embodiment as disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A to FIG. 1D show schematic illustrations of how a Type I and TypeII membrane protein (FIG. 1A and FIG. 1C) may be engineered withtransmembrane and intracellular domains removed and adjoined using alinker sequence (FIG. 1B) to generate a single chimeric protein whereinthe extracellular domains of the Type I and Type II membrane proteinseach face outward in a single chimeric protein. FIG. 1B depicts thelinkage of a Type I and Type II membrane protein by removal of thetransmembrane and intracellular domains of each protein, and where theliberated extracellular domains (ECD) from each protein have beenadjoined by a linker sequence. The ECD in this depiction may include theentire amino acid sequence of a candidate Type I or Type II proteinwhich is typically localized outside the cell membrane, or any portionthereof which retains binding to the intended receptor or ligand. FIG.1D depicts adjoined extracellular domains in a linear construct whereinthe extracellular domain of the Type I membrane protein faces the “left”side of the construct and the extracellular domain of the Type IImembrane protein faces the “right” side of the construct.

FIG. 2 shows, without wishing to be bound by theory, an in silicopredicted structure of a monomeric CD172a (SIRPα)-Fc-CD40L chimericprotein (SL-172154).

FIG. 3A shows, without wishing to be bound by theory, a schematicdiagram illustrating a mechanism of action of the hCD172a(SIRPα)-Fc-CD40L chimeric protein for stimulating active tumordestruction. The chimeric protein may then “dangle” from the surface ofthe tumor cell, and the CD40L portion of the chimeric protein may thenbind to CD40 expressed on the surface of the T cell. This would resultin replacement of an inhibitory hCD172a (SIRPα) signal with aco-stimulatory CD40L signal to enhance the anti-tumor activity of Tcells. FIG. 3B shows a synapse that has formed by a chimeric proteinbetween a tumor cell and a T cell.

FIG. 4 shows characterization of human CD172a (SIRPα)-Fc-CD40L chimericprotein (SL-172154) by Western blot. Specifically, each individualdomain of the chimeric protein was probed using an anti-CD172a, anti-Fc,or anti-CD40L antibody. Untreated samples of the hCD172a(SIRPα)-Fc-CD40L chimeric protein, e.g., control, were loaded into lane2 in all the blots (no 3-mercaptoethanol or PNGase). Samples in lane 3were treated with the reducing agent, 3-mercaptoethanol, while samplesin lane 4 were treated with PNGase.

FIG. 5A to FIG. 5C are tables of results showing the identified bindingpartners of human PD1-Fc-OX40L (FIG. 5A), of human CSF1R-Fc-CD40L (FIG.5B), or of human CD172a (SIRPα)-Fc-CD40L (FIG. 5C) from a microarraycontaining about 6,000 human membrane proteins. In each case, theexpected binding partners for each candidate molecule were identified bythe screen. There was no evidence of non-specific binding to other humanproteins, and binding to Galectin-1 is seen in the screen for allFc-containing fusion proteins.

FIG. 6 shows ELISA assays demonstrating the binding affinity of thedifferent domains of human CD172a (SIRPα)-Fc-CD40L chimeric protein fortheir respective binding partners. The first (left-most) panel shows thebinding and detection of hCD172a (SIRPα)-Fc-CD40L chimeric protein tohuman IgG, the binding partner for Fc. Human Ig (hIg) was used as astandard. The second panel from the left shows the binding and detectionof hCD172a (SIRPα)-Fc-CD40L chimeric protein to CD47, the bindingpartner for CD172a. The third panel from the left shows the binding anddetection of hCD172a (SIRPα)-Fc-CD40L chimeric protein to the receptorCD40, the binding partner for CD40L. The last (right-most) paneldemonstrates a dual-binding functional ELISA assay where recombinanthuman CD40 was used to capture the CD40L domain and recombinant humanCD47 was used to detect the CD172a (SIRPα) domain, demonstratingcontemporaneous binding of CD172a (SIRPα)-Fc-CD40L to both of itsbinding partners.

FIG. 7A and FIG. 7B show ex vivo cell binding assays demonstrating theability of different domains of the CD172a (SIRPα)-Fc-CD40L chimericprotein to bind their respectively binding partners (e.g., receptor orligand) on the surface of a mammalian cell membrane. FIG. 7A showsbinding of the human CD172a (SIRPα)-Fc-CD40L chimeric protein to hCD47(top curve is HeLa/hCD47, bottom is HeLa Parental). FIG. 7B showsbinding of the murine CD172a (SIRPα)-Fc-CD40L chimeric protein to mCD40(top curve is CHOK1/mCD40, bottom curve is CHOK1 Parental).

FIG. 8A to FIG. 8E show the binding affinity of the human CD172a(SIRPα)-Fc-CD40L chimeric protein by surface plasmon resonance (SPR).FIG. 8A shows binding of the hCD172a (SIRPα)-Fc-CD40L chimeric proteinto hCD47 (top curve is CD172A-Fc-CD40L (250 nM), bottom curve isCD172A-Fc-Cntl (250 nM)). FIG. 8B shows binding of the hCD172a(SIRPα)-Fc-CD40L chimeric protein to hFcγR1A (bottom curve isCD172a-Fc-CD40L (250 nM), top curve is CD172a-Fc-Cntl (250 nM)). FIG. 8Cshows binding of the hCD172a (SIRPα)-Fc-CD40L chimeric protein to hFcRn(bottom curve is CD172a-Fc-CD40L (250 nM), top curve is CD172A-Fc-Cntl(250 nM)). FIG. 8D summarizes the affinity results in FIG. 8A to FIG.8C. FIG. 8E shows binding affinity of the murine CD172a (SIRPα)-Fc-CD40Lchimeric protein to mCD40.

FIG. 9A to FIG. 9C show ex vivo functional assays of the human CD172a(SIRPα)-Fc-CD40L chimeric protein. FIG. 9A shows an ELISA-based blockingassay demonstrating the binding of hCD172a (SIRPα)-Fc-CD40L to cellsoverexpressing hCD47. FIG. 9B shows a schematic representation of themode of action of a macrophage engulfment assay. FIG. 9C shows increasedlevels of double positive cells (phagocytosis) in response to CD172a(SIRPα)-Fc-CD40L chimeric protein in a concentration dependent manner.

FIG. 10A and FIG. 10B show characterization of murine CD172a(SIRPα)-Fc-CD40L chimeric protein by Western blot analysis and ELISAassays. FIG. 10A shows the detection of each individual domain of themCD172a (SIRPα)-Fc-CD40L fusion construct using an anti-CD172a, anti-Fc,or anti-CD40L antibody. Untreated samples of the mCD172a(SIRPα)-Fc-CD40L chimeric protein, e.g., control, were loaded into lane2 in all the blots (no β-mercaptoethanol or PNGase). Samples in lane 3were treated with the reducing agent, 3-mercaptoethanol, while samplesin lane 4 were treated with PNGase. FIG. 10B shows ELISA assays todemonstrate the binding affinity of the different domains of mCD172a(SIRPα)-Fc-CD40L chimeric protein for their respective binding partners.In binding to CD47 (FIG. 10B, left side) the binding and detection ofthe mCD172a (SIRPα)-Fc-CD40L chimeric protein to CD47, the bindingpartner for CD172a (square symbols) were demonstrated. RecombinantmCD172a-mFc was used to generate a standard curve (circle symbols). Inbinding to CD40 (FIG. 10B, right side) the binding and detection of themCD172a (SIRPα)-Fc-CD40L chimeric protein to the receptor CD40, thebinding partner for CD40L (square symbols) were demonstrated.Recombinant mCD40L was used to generate a standard curve (circlesymbols).

FIG. 11A to FIG. 11C shows results from in vivo tumor studiesdemonstrating the anti-tumor efficacy of mCD172a (SIRPα)-Fc-CD40Lchimeric protein. A CT26 tumor was implanted into Balb/c mice prior totreatment with anti-CD47, anti-CD40, a combination of the twoantibodies, with mCD172a (SIRPα)-Fc-CD40L, or with control antibodies.FIG. 11A shows the evolution of tumor size over forty-five days aftertumor inoculation for each group.

FIG. 11B shows the overall survival percentage of mice through fiftydays after tumor inoculation (at day 50, the curves top to bottom are:CD172a-Fc-CD40L (150 μg×2), CD172a-Fc-CD40L (300 μg×2), αCD47/CD40,αCD40, αCD47 (0% survival by between days 30-35) and untreated (0%survival by between days 20-25). FIG. 11C summarizes the group sizes andtreatment outcomes for each group.

FIG. 12A to FIG. 12F show in vivo functional assays of the mCD172a(SIRPα)-Fc-CD40L chimeric protein. Immune profiling was performed ontumor-bearing mice treated with the mCD172a (SIRPα)-Fc-CD40L chimericprotein. FIG. 12A shows changes in the CD4+ and CD8+ T-cell populationsin the spleen in mice treated with the chimeric protein. FIG. 12B showschanges in the CD4+CD25− effector T cells or CD4+CD25+ regulatory Tcells in the spleen of mice treated with the chimeric protein. FIG. 12Cand FIG. 12D show changes in the CD4+ and CD8+ T-cell populations in theperipheral lymph nodes and tumor, respectively, in mice treated with thechimeric protein. FIG. 12E shows tetramer staining for determining thefraction of CD8+ T cells within splenocytes or tumor infiltratedlymphocytes (TIL) that recognized the AH1 tumor antigen nativelyexpressed by CT26 tumors. FIG. 12F shows changes in the proportion ofcells which upregulate IL-15 receptor alpha (IL15Rα), which is anindicator of CD40 activation. In each of FIG. 12A to FIG. 12F, theconditions from left to right are: untreated, αCD40/CD47, mCD172a(SIRPα)-Fc-CD40L (150 μg×2), mCD172a (SIRPα)-Fc-CD40L (300 μg×2).

FIG. 13 shows data from cynomolgus macaques treated with human CD172a(SIRPα)-Fc-CD40L. 1 male and 1 female cynomolgus macaque were treatedwith a single dose of hCD172a (SIRPα)-Fc-CD40L at 1 mg/kg. Serum wascollected at multiple time points from pre-treatment to fourteen daysafter treatment to evaluate pharmacokinetics and safety. CBC/CMPs wereperformed at five time points for safety and specific evaluation ofhemolysis and thrombocytopenia. No evidence of red blood cell lysis orplatelet depletion were observed following treatment with hCD172a(SIRPα)-Fc-CD40L. Gross safety assessments were made multiple timesdaily, and no additional safety signals were observed.

FIG. 14 shows in vivo synergy in reducing tumor volumes of treatmentswith antibodies (as monotherapies or in combinations) directed tocheckpoint proteins or treatments with chimeric proteins. The conditionsare, left to right: vehicle, anti-OX40 (OX86), anti-PD-1 (RMP1-14),anti-PD-1 (29F.1A12), anti-TIM3 (RMT3-23), anti-TIM3+OX40,anti-TIM3+OX40+PD-1 (RMP1-14), anti-TIM3+OX40+PD-1 (29F.1A12), ARC300ug×2 (“ARC” is the TIM3-Fc-OX40L chimeric protein).

FIG. 15A shows in vivo reduction in tumor volume from sequentialtreatments of chimeric proteins, either two sequential treatments withthe same chimeric protein or treatments with two different chimericproteins. FIG. 15B shows percent survival over time for the mice shownin FIG. 15A. For clarity, in FIG. 15A, the conditions at point 20 dayson the x axis are (top to bottom): vehicle,CSF1R-Fc-CD40L—TIM3-Fc-OX40L, TIM3-Fc-OX40L (150 μg×2),TIM3-Fc-OX40L—CSF1R-Fc-CD40L, TIM3-Fc-OX40L—CD172a-Fc-CD40L, andCD172a-Fc-CD40L—TIM3-Fc-OX40L. For clarity, in FIG. 15B, the conditionsare identified as: vehicle: “1”; TIM3-Fc-OX40L (150 μg×2): “2”;TIM3-Fc-OX40L—CD172a-Fc-CD40L: “3”; TIM3-Fc-OX40L—CSF1R-Fc-CD40L: “4”;CD172a-Fc-CD40L—TIM3-Fc-OX40L: “5”; and CSF1R-Fc-CD40L—TIM3-Fc-OX40L:“6”.

FIG. 16 shows, without wishing to be bound by theory, four potentialconfigurations of PD-1-Fc-OX40L chimeric proteins.

FIG. 17 shows Western blots of PD-1-Fc-OX40L chimeric proteins run onSDS-PAGE under a non-reducing condition, a reducing condition, and areducing condition and following treatment with Peptide-N-Glycosidase F(PNGaseF).

FIG. 18 shows a chromatograph for PD-1-Fc-OX40L chimeric proteins run onSize Exclusion Chromatography (SEC).

FIG. 19 shows SDS-PAGE and native (non-SDS) PAGE gels for PD-1-Fc-OX40Lchimeric proteins run under a non-reducing condition (“−”) or a reducingcondition (“+”).

FIG. 20 shows a native (non-SDS) PAGE gel for PD-1-No Fc-OX40L chimericproteins which lack a Fc domain.

FIG. 21 shows, without wishing to be bound by theory, a model for how ahexamer and concatemers form from chimeric proteins of the presentinvention.

FIG. 22A to FIG. 22Q show characterization of PD-1-Fc-OX40L chimericproteins with different joining linker sequences by Western blotanalysis. Sequences of the different joining linkers are provided belowin the Examples section. Specifically, each individual domain of thefusion construct was probed using an α-PD-1, α-Fc, or α-OX40L antibody.In each figure, untreated samples of the PD-1-Fc-OX40L chimeric protein,e.g., control, were loaded into lane 1 in all the blots (noβ-mercaptoethanol or PNGase). Samples in lane 2 were treated with thereducing agent, β-mercaptoethanol, while samples in lane 3 were treatedwith PNGase.

FIG. 23 shows characterization of PD-1-Fc-OX40L chimeric proteins withdifferent joining linker sequences by ELISA-based capture and detectionassay against the central Fc region of the protein. The proteinconcentration of each PD-1-Fc-OX40L chimeric protein with differentjoining linker sequence (#1 to #17) was determined.

FIG. 24A to FIG. 24P show the flow cytometry profiles of PD-1-Fc-OX40Lchimeric proteins with different joining linker sequences by FACSanalysis to PD-L1 or OX40. The EC₅₀ values were calculated for eachPD-1-Fc-OX40L chimeric protein with different joining linker sequence(#2 to #17).

FIG. 25A, using an PD1-Fc-OX40L chimeric protein as a non-limitingexample, shows that tumor cells may express PD-L1 on the cell surface,which can bind to PD-1 expressed by a T cell (FIG. 25B). Thisinteraction suppresses activation of T cells. A chimeric proteincomprising the extracellular domain of PD-1, adjoined to theextracellular domain of OX40L may bind to PD-L1 on the surface of atumor cell, preventing binding to PD-1 on the surface of a T cell (FIG.25C). The chimeric protein may then “dangle” from the surface of thetumor cell, and the OX40L portion of the chimeric protein may then bindto OX40 expressed on the surface of the T cell. This would result inreplacement of an inhibitory PD-L1 signal with a co-stimulatory OX40Lsignal to enhance the anti-tumor activity of T cells.

FIG. 26A shows cell lines generated to overexpress murine PD-L1(CHOK1/mPD-L1), PD-L2 (CHOK1/mPD-L2), or OX40 (CHOK1/mOX40). FIG. 26Bshows in vitro cell binding to demonstrate the ability of the murinePD-1-Fc-OX40L chimeric protein to bind to the engineered cell lines.FIG. 26C and FIG. 26D show Staphylococcus aureas, Enterotoxin Type B(SEB) superantigen cytokine release assays which demonstrate the effectsof the mPD-1-Fc-OX40L chimeric protein on IL-2 (FIG. 26C) and TNFα (FIG.26D) secretion. In FIG. 26C and FIG. 26D, curves top to bottom at x axispoint 2.5 are: PD1-Fc-OX40L, PD1-Fc/OX40L, OX40L-Fc, PD1-Fc/OX40L-Fc,PD1-Fc, and IgG control.

FIG. 27A shows the evolution of in vivo tumor size after CT26 tumorinoculation for each group of mice described in the figure. FIG. 27B andFIG. 27C show the overall survival percentage, and statistics, of miceand tumor rejection through forty days after tumor inoculation. In FIG.27B, the different treatment conditions are identified as: untreated:“a”, “e”, and “h”; αPD-L1 (10F.9G2): “b”; αPD-1 (RMP1-14): “c”; αOX40(OX86): “d”; PD-L1/OX40: “f”; αPD-1 (RMP1-14)/OX40: “g”; PD-1-Fc-OX40L(100 μg×2): “i”; PD-1-Fc-OX40L (150 μg×2): “j”; and PD-1-Fc-OX40L (300μg×2): “k”. FIG. 27D and FIG. 27E show immune profiling performed onCT26-tumor bearing mice treated with the murine PD-1-Fc-OX40L chimericprotein or antibodies (as monotherapies of αPD-1, α PD-L1, or αOX40 oras combination therapy of αPD-L1 and αOX40 or αPD-1 and αOX40). In bothFIG. 27D and FIG. 27E, the order of test articles from left to right is:untreated, αPD-1 (RMP1-14), αPD-L1 (10F.9G2), αOX40 (OX86), αPD-1(RMP1-14)/OX40, PD-L1/OX40, PD-1-Fc-OX40L (150 μg×2), and PD-1-Fc-OX40L(300 μg×2). FIG. 27F summarizes the treatment outcomes for eachexperimental group. FIG. 27G and FIG. 27H show in vivo anti-tumoractivity of the mPD-1-Fc-OX40L chimeric protein in the CT26 tumor model.In FIG. 27G, left two panels: untreated is the top curve. In FIG. 27G,third panel from left: curves top to bottom are untreated, αPD1 (2×100μg), αPD-L1 (1×100 μg), and αPD-L1 (2×100 μg). In FIG. 27G, rightmostpanel: curves top to bottom are untreated, αOX40/αPD-L1 (1×100 μg),αOX40/αPD-L1 (2×100 μg), αOX40/αPD1 (2×100 μg). In FIG. 27H, panellabelled “ARC Fusion Protein,” curves are, top to bottom: untreated,PD1-Fc-OX40L (300 ug×2), and PD1-Fc-OX40L (150 ug×2). In FIG. 27H, panellabelled “OX40 Agonist,” curves are, top to bottom: untreated and αOX86.In FIG. 27H, panel labelled “PD-1/L1 Blockade,” curves are, top tobottom: untreated, αPD1, αPD-L1. In FIG. 27H, panel labelled “AntibodyCombinations,” curves are, top to bottom: untreated, αPD1/αOX86,αPD-L1/αOX86.

FIG. 28A to FIG. 28C show ELISA assays demonstrating binding affinity ofthe different domains of human PD-1-Fc-OX40L chimeric protein (alsoreferred to as SL-279252) for their respective binding histogramspartners. In FIG. 28C, for each concentration, left is OX40-His andright is HVEM-His.

FIG. 29A shows the characterization of human cell lines used for invitro binding to human PD-1-Fc-OX40L. FIG. 29B to FIG. 29D show bindingof hPD-1-Fc-OX40L to cells expressing, respectively, PD-L1, PD-L2, orOX40. In each, the right panel shows the titration curve for increasingconcentrations of the chimeric protein.

FIG. 30 shows characterization of human PD-1-Fc-OX40L fusion protein(SL-279252) by Western blot analysis. Each of the three domains of thechimeric protein was probed, respectively, with an anti-PD-1, anti-Fc,or anti-OX40L antibody. Untreated samples of the hPD-1-Fc-OX40L fusionprotein, e.g., control, were loaded into lane 1 in all the blots (noβ-mercaptoethanol or Peptide:N-Glycosidase (PNGase).

FIG. 31A and FIG. 31B show functional ELISA (FIG. 31A) and cell bindingassays (FIG. 31B) which determine whether glycosylation of thePD-1-Fc-OX40L fusion protein (SL-279252) impacts its function.

FIG. 32A to 32G show in vitro functional assays of human PD-1-Fc-OX40Lfusion protein. In FIG. 32C, the hPD-1-Fc-OX40L induced higher levels ofsecreted 1L2 in PC3 cells (FIG. 32C, left bundle) than in HCC827 cells(FIG. 32C, right bundle). FIG. 32D is a flow cytometry analysis of cellstaken from the T cell/tumor cell co-culture assays outlined in FIG. 32C.The left-most bars indicate the proportion of CD4+ or CD8+ cellsexpressing Ki67 (as an indicator of proliferation) in the absence oftumor cells. The second from left bars indicates the proportion of CD4+or CD8+ cells expressing Ki67 in the presence of tumor cells but withoutARC, whereas the third-from left and right-most bars indicate theproportion of CD4+ or CD8+ cells expressing Ki67 in the presence of 500ng or 5 ug of ARC, respectively. FIG. 32F and FIG. 32G, the order oftest articles in the inset histograms, left to right: media control, IgGcontrol, PD1-Fc, OX40L-Fc, PD-1-Fc/OX40L-Fc, and PD-1-Fc-Ox40L.

FIG. 33A to FIG. 33C show ELISA assays demonstrating binding affinityand cross-reactivity of the human PD-1-Fc-OX40L fusion protein to PD-L1(FIG. 33A) and PD-L2 (FIG. 33A) of cynomolgus macaque or to OX40 (FIG.33C) of rhesus macaque.

FIG. 34 shows, without wishing to be bound by theory, an in silicopredicted structure of monomeric TIM3-Fc-OX40L chimeric protein(SL-366252).

FIG. 35 shows characterization of murine TIM3-Fc-OX40L chimeric protein(SL-366252) by Western blot analysis. Specifically, each individualdomain of the fusion construct was probed using an anti-TIM3, anti-Fc,or anti-OX40L antibody. Untreated samples of the mTIM3-Fc-OX40L chimericprotein, e.g., control, were loaded into lane 2 in all the blots (noβ-mercaptoethanol or PNGase). Samples in lane 3 were treated with thereducing agent, 3-mercaptoethanol, while samples in lane 4 were treatedwith PNGase.

FIG. 36 shows ex vivo cell binding assays demonstrating the ability ofthe TIM3-Fc-OX40L chimeric protein to bind its binding partners (e.g.,receptor or ligand) on the surface of a mammalian cell membrane.Specifically, the graph shows the binding of the TIM3-Fc-OX40L chimericprotein to mOX40 (CHOK1-mOX40 is top curve, CHOK1 parental is bottom).

FIG. 37A to FIG. 37C show in vivo functional assays of the murineTIM3-Fc-OX40L chimeric protein. Immune profiling was performed ontumor-bearing mice treated with mTIM3-Fc-OX40L chimeric protein. FIG.37A shows changes in the CD4+ and CD8+ T-cell populations in micetreated with the chimeric protein. FIG. 37B shows changes in theCD4+CD25− effector T cells or CD4+CD25+ regulatory T cells in micetreated with the chimeric protein. FIG. 37C shows tetramer staining toanalyze the fraction of CD8+ T cells within splenocytes or tumorinfiltrated lymphocytes (TIL) that recognized the AH1 tumor antigennatively expressed by CT26 tumors. In all of the panels, the leftcondition is untreated and the right condition is mTIM3-Fc-OX40L (150μg×2).

FIG. 38A to FIG. 38C show results from in vivo tumor studiesdemonstrating that mTIM3-Fc-OX40L chimeric protein had significantanti-tumor activity in the CT26 mouse model. Mice were inoculated withCT26 tumors. When tumors reached 4-5 mm, mice were treated twice with150 μg of mTIM3-Fc-OX40L chimeric protein or with control antibodies.FIG. 38A shows the evolution of tumor size over forty-five days aftertumor inoculation for each group.

FIG. 38B shows the overall survival percentage of mice through fiftydays after tumor inoculation (TIM3-Fc-OX40L is the top curve). FIG. 38Csummarizes the group sizes and treatment outcomes for each group.

FIG. 39 is a table showing joining linkers and Fc linkers that can becombined into exemplary modular linkers. The exemplary modular linkersshown can be combined with any herein-described Type I and Type IIproteins and/or extracellular domains of a herein described Type I andType II proteins to form a chimeric protein of the present invention.

DETAILED DESCRIPTION

The present invention is based, in part, on the discovery that chimericproteins can be engineered from the extracellular, or effector, regionsof immune-modulating transmembrane proteins in a manner that exploitsthe orientations of these proteins (e.g., Type I versus Type II) andtherefore allows the delivery of immune stimulatory and/or immuneinhibitory signals, including, for example, masking an immune inhibitorysignal and replacing it with an immune stimulatory signal in thetreatment of cancer.

Chimeric Proteins

In some aspects, the chimeric protein is of a general structure of: Nterminus-(a)-(b)-(c)-C terminus, where (a) is a first domain comprisingan extracellular domain of a Type I transmembrane protein, (b) is alinker having at least one cysteine residue capable of forming adisulfide bond (including without limitation, hinge-CH2-CH3 Fc domain isderived from human IgG4), and (c) is a second domain comprising anextracellular domain of Type II transmembrane protein, where the linkerconnects the first domain and the second domain and optionally comprisesone or more joining linkers as described herein, where one of the firstand second extracellular domains is an immune inhibitory signal and oneof the first and second extracellular domains is an immune stimulatorysignal.

In embodiments, chimeric protein refers to a recombinant fusion protein,e.g., a single polypeptide having the extracellular domains describedherein. For example, in embodiments, the chimeric protein is translatedas a single unit in a cell. In embodiments, chimeric protein refers to arecombinant protein of multiple polypeptides, e.g., multipleextracellular domains described herein, that are linked to yield asingle unit, e.g., in vitro (e.g., with one or more synthetic linkersdescribed herein). In embodiments, the chimeric protein is chemicallysynthesized as one polypeptide or each domain may be chemicallysynthesized separately and then combined. In embodiments, a portion ofthe chimeric protein is translated and a portion is chemicallysynthesized.

In embodiments, an extracellular domain refers to a portion of atransmembrane protein which is capable of interacting with theextracellular environment. In embodiments, an extracellular domainrefers to a portion of a transmembrane protein which is sufficient tobind to a ligand or receptor and effective transmit a signal to a cell.In embodiments, an extracellular domain is the entire amino acidsequence of a transmembrane protein which is external of a cell or thecell membrane. In embodiments, an extracellular domain is the thatportion of an amino acid sequence of a transmembrane protein which isexternal of a cell or the cell membrane and is needed for signaltransduction and/or ligand binding as may be assayed using methods knowin the art (e.g., in vitro ligand binding and/or cellular activationassays).

In embodiments, an immune inhibitory signal refers to a signal thatdiminishes or eliminates an immune response. For example, in the contextof oncology, such signals may diminish or eliminate antitumor immunity.Under normal physiological conditions, inhibitory signals are useful inthe maintenance of self-tolerance (e.g., prevention of autoimmunity) andalso to protect tissues from damage when the immune system is respondingto pathogenic infection. For instance, without limitation, immuneinhibitory signal may be identified by detecting an increase in cellularproliferation, cytokine production, cell killing activity or phagocyticactivity when such an inhibitory signal is blocked.

In embodiments, an immune stimulatory signal refers to a signal thatenhances an immune response. For example, in the context of oncology,such signals may enhance antitumor immunity. For instance, withoutlimitation, immune stimulatory signal may be identified by directlystimulating proliferation, cytokine production, killing activity orphagocytic activity of leukocytes. Specific examples include directstimulation of TNF superfamily receptors such as OX40, LTbR, 4-1BB orTNFRSF25 using either receptor agonist antibodies or using chimericproteins encoding the ligands for such receptors (OX40L, LIGHT, 4-1BBL,TL1A, respectively). Stimulation from any one of these receptors maydirectly stimulate the proliferation and cytokine production ofindividual T cell subsets. Another example includes direct stimulationof an immune inhibitory cell with through a receptor that inhibits theactivity of such an immune suppressor cell. This would include, forexample, stimulation of CD4+FoxP3+ regulatory T cells with a GITRagonist antibody or GITRL containing chimeric protein, which wouldreduce the ability of those regulatory T cells to suppress theproliferation of conventional CD4+ or CD8+ T cells. In another example,this would include stimulation of CD40 on the surface of an antigenpresenting cell using a CD40 agonist antibody or a chimeric proteincontaining CD40L, causing activation of antigen presenting cellsincluding enhanced ability of those cells to present antigen in thecontext of appropriate native costimulatory molecules, including thosein the B7 or TNF superfamily. In another example, this would includestimulation of LTBR on the surface of a lymphoid or stromal cell using aLIGHT containing chimeric protein, causing activation of the lymphoidcell and/or production of pro-inflammatory cytokines or chemokines tofurther stimulate an immune response, optionally within a tumor.

Membrane proteins typically consist of an extracellular domain, one or aseries of trans-membrane domains, and an intracellular domain. Withoutwishing to be bound by theory, the extracellular domain of a membraneprotein is responsible for interacting with a soluble or membrane boundreceptor or ligand. Without wishing to be bound by theory, thetrans-membrane domain(s) are responsible for localizing a protein to theplasma membrane. Without wishing to be bound by theory, theintracellular domain of a membrane protein is responsible forcoordinating interactions with cellular signaling molecules tocoordinate intracellular responses with the extracellular environment(or visa-versa). There are two types of single-pass membrane proteins,those with an extracellular amino terminus and intracellular carboxyterminus (Type I) and those with an extracellular carboxy terminus andintracellular amino terminus (Type II). Both Type I and Type II membraneproteins can be either receptors or ligands. For Type I membraneproteins, the amino terminus of the protein faces outside the cell, andtherefore contains the functional domains that are responsible forinteracting with other binding partners (either ligands or receptors) inthe extracellular environment. For Type II membrane proteins, thecarboxy terminus of the protein faces outside the cell, and thereforecontains the functional domains that are responsible for interactingwith other binding partners (either ligands or receptors) in theextracellular environment. Thus, these two types of proteins haveopposite orientations to each other.

Because the outward facing domains of Type I and Type II membraneproteins are opposite, it is possible to link the extracellular domainsof a Type I and Type II membrane protein such that the ‘outward facing’domains of the molecules are also in opposing orientation to each other(FIG. 1D). The resulting construct would therefore consist of theextracellular domain of a Type I membrane protein on the ‘left’ side ofthe molecule, connected to the extracellular domain of a Type IImembrane protein on the ‘right’ side of the molecule using a linkersequence. This construct could be produced by cloning of these threefragments (the extracellular domain of a Type I protein, followed by alinker sequence, followed by the extracellular domain of a Type IIprotein) into a vector (plasmid, viral or other) wherein the aminoterminus of the complete sequence corresponded to the ‘left’ side of themolecule containing the Type I protein and the carboxy terminus of thecomplete sequence corresponded to the ‘right’ side of the moleculecontaining the Type II protein. Accordingly, in embodiments, the presentchimeric proteins are engineered as such.

In embodiments, the extracellular domain may be used to produce asoluble protein to competitively inhibit signaling by that receptor'sligand. In embodiments, the extracellular domain may be used to provideartificial signaling.

In embodiments, the extracellular domain of a Type I transmembraneprotein is an immune inhibitory signal. In embodiments, theextracellular domain of a Type II transmembrane protein is an immunestimulatory signal.

In embodiments, the present chimeric proteins comprise an extracellulardomain of a Type I transmembrane protein, or a functional fragmentthereof. In embodiments, the present chimeric proteins comprise anextracellular domain of a Type II transmembrane protein, or a functionalfragment thereof. In embodiments, the present chimeric proteins comprisean extracellular domain of a Type I transmembrane protein, or afunctional fragment thereof, and an extracellular domain of a Type IItransmembrane protein, or a functional fragment thereof.

In embodiments, the present chimeric proteins may be engineered totarget one or more molecules that reside on human leukocytes including,without limitation, the extracellular domains (where applicable) ofSLAMF4, IL-2 R α, 4-1BB/TNFRSF9, IL-2 R β, ALCAM, BTLA, B7-1, IL-4 R,B7-H3, BLAME/SLAMFS, CEACAM1, IL-6 R, IL-7 Rα, IL-10R α, IL-I 0 R β,IL-12 R β 1, IL-12 R β 2, CD2, IL-13 R α 1, IL-13, CD3, CD4, ILT2/CDS5j,ILT3/CDS5k, ILT4/CDS5d, ILT5/CDS5a, lutegrin α 4/CD49d, CDS, Integrin αE/CD103, CD6, Integrin α M/CD 11 b, CDS, Integrin α X/CD11c, Integrin β2/CDIS, KIR/CD15S, CD27/TNFRSF7, KIR2DL1, CD2S, KIR2DL3, CD30/TNFRSFS,KIR2DL4/CD15Sd, CD31/PECAM-1, KIR2DS4, CD40 Ligand/TNFSF5, LAG-3, CD43,LAIR1, CD45, LAIR2, CDS3, Leukotriene B4-R1, CDS4/SLAMF5, NCAM-L1, CD94,NKG2A, CD97, NKG2C, CD229/SLAMF3, NKG2D, CD2F-10/SLAMF9, NT-4, CD69,NTB-A/SLAMF6, Common γ Chain/IL-2 R γ, Osteopontin, CRACC/SLAMF7, PD-1,CRTAM, PSGL-1, CTLA-4, RANK/TNFRSF11A, CX3CR1, CX3CL1, L-Selectin, SIRPβ 1, SLAM, TCCR/WSX-1, DNAM-1, Thymopoietin, EMMPRIN/CD147, TIM-1,EphB6, TIM-2, Fas/TNFRSF6, TIM-3, Fas Ligand/TNFSF6, TIM-4, FcγRIII/CD16, TIM-6, TNFR1/TNFRSF1A, Granulysin, TNF RIII/TNFRSF1B, TRAILRI/TNFRSFIOA, ICAM-1/CD54, TRAIL R2/TNFRSF10B, ICAM-2/CD102,TRAILR3/TNFRSF10C, IFN-γR1, TRAILR4/TNFRSF10D, IFN-γ R2, TSLP, IL-1 R1,LIGHT, LTBR (TNFRSF3) and TSLP R.

The activation of regulatory T cells is critically influenced bycostimulatory and coinhibitory signals. Two major families ofcostimulatory molecules include the B7 and the tumor necrosis factor(TNF) families. These molecules bind to receptors on T cells belongingto the CD28 or TNF receptor families, respectively. Many well-definedcoinhibitors and their receptors belong to the B7 and CD28 families.

In embodiments, the present chimeric proteins may be engineered totarget one or more molecules involved in immune inhibition, includingfor example: CSF1R, CTLA-4, PD-L1, PD-L2, PD-1, BTLA, HVEM, TIM3, GAL9,VISTA/VSIG8, KIR, 2B4, TIGIT, CD160 (also referred to as BY55), CHK 1and CHK2 kinases, A2aR, CEACAM (e.g., CEACAM-1, CEACAM-3 and/orCEACAM-5), and various B-7 family ligands (including, but are notlimited to, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6and B7-H7).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of an immune inhibitory agent, including withoutlimitation, one or more of TIM-3, BTLA, PD-1, CSF1R, CTLA-4, CD244,CD160, TIGIT, CD172a (SIRP1α), 2B4, VISTA, VSIG8, CD200 and TMIGD2.

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of a Type I membrane protein which has immuneinhibitory properties. In embodiments, the chimeric protein isengineered to disrupt, block, reduce, and/or inhibit the transmission ofan immune inhibitory signal.

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of an immune stimulatory signal is one or moreof 4-1BBL, OX-40 ligand (OX-40L), LIGHT (CD258), GITR ligand (GITRL),CD70, CD30 ligand, CD40 ligand (CD40L), CD137 ligand, TRAIL, and TL1A.

In embodiments, the chimeric protein simulates binding of an inhibitorysignal ligand to its cognate receptor (e.g., PD-1 to PD-L1 or PD-L2;e.g., CD172a (SIRP1α) to CD47; e.g., CD115 to CSF1; e.g., TIM-3 togalectin-9 or phosphatidylserine) but inhibits the inhibitory signaltransmission to an immune cell (e.g., a T cell, macrophage or otherleukocyte).

In embodiments, the chimeric protein comprises an immune inhibitoryreceptor extracellular domain and an immune stimulatory ligandextracellular domain which can, without limitation, deliver an immunestimulation to a T cell while masking a tumor cell's immune inhibitorysignals. In embodiments, the chimeric protein delivers a signal that hasthe net result of T cell activation.

In embodiments, the chimeric protein comprises an immune inhibitorysignal which is an ECD of a receptor of an immune inhibitory signal andthis acts on a tumor cell that bears a cognate ligand of the immuneinhibitory signal. In embodiments, the chimeric protein comprises animmune stimulatory signal which is an ECD of a ligand of an immunestimulatory signal and this acts on a T cell that bears a cognatereceptor of the immune stimulatory signal. In embodiments, the chimericprotein comprises both (i) an immune inhibitory signal which is areceptor of an immune inhibitory signal and this acts on a tumor cellthat bears a cognate ligand of the immune inhibitory signal and (ii) animmune stimulatory signal which is a ligand of an immune stimulatorysignal and this acts on a T cell that bears a cognate receptor of theimmune stimulatory signal.

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of one or more of the immune-modulating agentsdescribed in Mahoney, Nature Reviews Drug Discovery 2015:14; 561-585,the entire contents of which are hereby incorporated by reference.

In embodiments, a chimeric protein is capable of binding murineligand(s)/receptor(s).

In embodiments, a chimeric protein is capable of binding humanligand(s)/receptor(s)

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of a Type II membrane protein which has immunestimulatory properties. In embodiments, the chimeric protein isengineered to enhance, increase, and/or stimulate the transmission of animmune stimulatory signal.

In embodiments, the chimeric protein comprises the extracellular domainof the immune inhibitory agent PD-1 and is paired with an immunestimulatory agent as follows: PD-1/4-1BBL; PD-1/OX-40L; PD-1/LIGHT;PD-1/GITRL; PD-1/CD70; PD-1/CD30L; PD-1/CD40L; and PD-1/TL1A. Inembodiments the chimeric protein is PD-1-Fc-LIGHT or PD-1-Fc-OX40L, inwhich the Fc represents a linker that comprises at least a portion of anFc domain of an antibody and which comprises at least one cysteineresidue capable of forming a disulfide bond.

In an embodiment, the chimeric protein comprises the extracellulardomain of the immune inhibitory agent PD-1 and is paired with the immunestimulatory agent OX-40L. In embodiments, the chimeric protein binds tohuman PD-L1 or PD-L2 with a K_(D) of about 1 nM to about 5 nM, forexample, about 1 nM, about 1.5 nM, about 2 nM, about 2.5 nM, about 3 nM,about 3.5 nM, about 4 nM, about 4.5 nM, or about 5 nM. In embodiments,the chimeric protein binds to human PD-L1 with a K_(D) of about 5 nM toabout 15 nM, for example, about 5 nM, about 5.5 nM, about 6 nM, about6.5 nM, about 7 nM, about 7.5 nM, about 8 nM, about 8.5 nM, about 9 nM,about 9.5 nM, about 10 nM, about 10.5 nM, about 11 nM, about 11.5 nM,about 12 nM, about 12.5 nM, about 13 nM, about 13.5 nM, about 14 nM,about 14.5 nM, or about 15 nM.

In embodiments, the chimeric protein exhibits enhanced stability andprotein half-life. In embodiments, the chimeric protein binds to FcRnwith high affinity. In embodiments, the chimeric protein may bind toFcRn with a K_(D) of about 1 nM to about 80 nM. For example, thechimeric protein may bind to FcRn with a K_(D) of about 1 nM, about 2nM, about 3 nM, about 4 nM, about 5 nM, about 6 nM, about 7 nM, about 8nM, about 9 nM, about 10 nM, about 15 nM, about 20 nM, about 25 nM,about 30 nM, about 35 nM, about 40 nM, about 45 nM, about 50 nM, about55 nM, about 60 nM, about 65 nM, about 70 nM, about 71 nM, about 72 nM,about 73 nM, about 74 nM, about 75 nM, about 76 nM, about 77 nM, about78 nM, about 79 nM, or about 80 nM. In an embodiment, the chimericprotein may bind to FcRn with a K_(D) of about 9 nM. In embodiments, thechimeric protein does not substantially bind to other Fc receptors (i.e.other than FcRn) with effector function.

In embodiments, the chimeric protein comprises the extracellular domainof the immune inhibitory agent PD-L1 or PD-L2 and is paired with animmune stimulatory receptor as follows: PD-L1/4-1BB; PD-L1/OX-40;PD-L1/HVEM; PD-L1/GITR; PD-L1/CD27; PD-L1/CD28; PD-L1/CD30; PD-L1/CD40and PD-L1/CD137.

In embodiments, the chimeric protein comprises the extracellular domainof the immune inhibitory agent PD-L2 and is paired with an immunestimulatory receptor as follows: PD-L2/4-1BB; PD-L2/OX-40; PD-L2/HVEM;PD-L2/GITR; PD-L2/CD27; PD-L2/CD28; PD-L2/CD30; PD-L2/CD40 andPD-L2/CD137.

In embodiments, the chimeric protein comprises the extracellular domainof the immune inhibitory agent TIM-3 and is paired with an immunestimulatory agent as follows: TIM-3/OX-40L; TIM-3/LIGHT; TIM-3/GITRL;TIM-3/CD70; TIM-3/CD30L; TIM-3/CD40L; TIM-3/CD137L; TIM-3/TL1A; andTIM-3/OX40L. In embodiments the chimeric protein is TIM3-Fc-OX40L, inwhich the Fc represents a linker that comprises at least a portion of anFc domain of an antibody and which comprises at least one cysteineresidue capable of forming a disulfide bond.

In embodiments, there is provided a method of treating a cancer or aninflammatory disease (e.g., any one of those described elsewhere herein)by administering to a subject a TIM3-Fc-OX40L chimeric protein, in whichthe Fc represents a linker that comprises at least a portion of an Fcdomain of an antibody and which comprises at least one cysteine residuecapable of forming a disulfide bond. In embodiments, the methodgenerates a memory response which may, e.g., be capable of preventingrelapse.

In embodiments, the chimeric protein comprises the extracellular domainof the immune inhibitory agent BTLA and is paired with an immunestimulatory agent as follows: BTLA/OX-40L; BTLA/LIGHT; BTLA/GITRL;BTLA/CD70; BTLA/CD30L; BTLA/CD40L; BTLA/CD137L; BTLA/TL1A; andBTLA/OX40L.

In embodiments, the chimeric protein comprises the extracellular domainof the immune inhibitory agent CD172a (SIRP1α) and is paired with animmune stimulatory agent as follows: CD172a (SIRP1α)/OX-40L; CD172a(SIRP1α)/LIGHT; CD172a (SIRP1α)/CD70; CD172a (SIRP1α)/CD30L; CD172a(SIRP1α)/CD40L; CD172a (SIRP1α)/CD137L; CD172a (SIRP1α)/TL1A; and CD172a(SIRP1α)/OX40L. In embodiments the chimeric protein is CD172a(SIRP1α)-Fc-CD40L or CD172a (SIRP1α)-Fc-LIGHT, in which the Fcrepresents a linker that comprises at least a portion of an Fc domain ofan antibody and which comprises at least one cysteine residue capable offorming a disulfide bond.

In embodiments, there is provided a method of treating a cancer or aninflammatory disease (e.g., any one of those described elsewhere herein)by administering to a subject a CD172a (SIRPα)-Fc-CD40L chimericprotein, in which the Fc represents a linker that comprises at least aportion of an Fc domain of an antibody and which comprises at least onecysteine residue capable of forming a disulfide bond. In embodiments,the method generates a memory response which may, e.g., be capable ofpreventing relapse. In embodiments, the method includes a sustainedtherapeutic effect of the CD172a (SIRPα)-Fc-CD40L, e.g., due to bindingof the extracellular domain components to their respective bindingpartners with slow off rates (K_(d) or K_(off)) to optionally providesustained negative signal masking effect and/or a longer positive signaleffect, e.g., to allow an effector cell to be adequately stimulated foran anti-tumor effect.

In embodiments, the chimeric protein comprises the extracellular domainof the immune inhibitory agent CD115 and is paired with an immunestimulatory agent as follows: CD115/OX-40L; CD115/LIGHT; CD115/CD70;CD115/CD30L; CD115/CD40L; CD115/CD137L; CD115/TL1A; and CD115/OX40L.

In embodiments, the chimeric protein comprises the extracellular domainof the immune inhibitory agent TMIGD2 and is paired with an immunestimulatory agent as follows: TMIGD2/OX-40L; TMIGD2/LIGHT; TMIGD2/GITRL;TMIGD2/CD70; TMIGD2/CD30L; TMIGD2/CD40L; TMIGD2/CD137L; TMIGD2/TL1A; andTMIGD2/OX40L.

In embodiments, the chimeric protein comprises the extracellular domainof the immune inhibitory agent CD200 and is paired with an immunestimulatory agent as follows: CD200/OX-40L; CD200/LIGHT; CD200/GITRL;CD200/CD70; CD200/CD30L; CD200/CD40L; CD200/CD137L; CD200/TL1A; andCD200/OX40L.

In embodiments, the present chimeric proteins may comprises variants ofthe extracellular domains described herein, for instance, a sequencehaving at least about 60%, or at least about 61%, or at least about 62%,or at least about 63%, or at least about 64%, or at least about 65%, orat least about 66%, or at least about 67%, or at least about 68%, or atleast about 69%, or at least about 70%, or at least about 71%, or atleast about 72%, or at least about 73%, or at least about 74%, or atleast about 75%, or at least about 76%, or at least about 77%, or atleast about 78%, or at least about 79%, or at least about 80%, or atleast about 81%, or at least about 82%, or at least about 83%, or atleast about 84%, or at least about 85%, or at least about 86%, or atleast about 87%, or at least about 88%, or at least about 89%, or atleast about 90%, or at least about 91%, or at least about 92%, or atleast about 93%, or at least about 94%, or at least about 95%, or atleast about 96%, or at least about 97%, or at least about 98%, or atleast about 99%) sequence identity with the known amino acid or nucleicacid sequence of the extracellular domains, e.g., human extracellulardomains, e.g., one or more of SEQ IDs NOs: 2, 4, 7, 10, 12, 15, 18, 21,24, 29, 32, 34, 36, 42, and 44.

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of TIM3 (SEQ ID NO: 2).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of PD-1 (SEQ ID NO: 7).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of CD172a (SIRP1α) (SEQ ID NO: 10).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of OX40L (SEQ ID NO: 4).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of CD40L (SEQ ID NO: 12).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of TIM3 (SEQ ID NO: 2) and the extracellulardomain of OX40L (SEQ ID NO: 4).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of PD-1 (SEQ ID NO: 7) and the extracellulardomain of OX40L (SEQ ID NO: 4).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of CD172a (SIRP1α) (SEQ ID NO: 10) and theextracellular domain of CD40L (SEQ ID NO: 12).

In embodiments, the chimeric protein of the present invention comprisesthe hinge-CH2-CH3 domain from a human IgG4 antibody sequence (SEQ ID NO:45, 46, or 47).

In embodiments, a chimeric protein comprises a modular linker as shownin FIG. 39.

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of TIM3 and the extracellular domain of OX40L,using the hinge-CH2-CH3 domain from a human IgG4 antibody sequence as alinker (this TIM3-Fc-OX40L chimera is SEQ ID NO: 5).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of PD-1 (SEQ ID NO: 7) and the extracellulardomain of OX40L, using the hinge-CH2-CH3 domain from a human IgG4antibody sequence as a linker (this PD-1-Fc-OX40L chimera is SEQ ID NO:8).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of CD172a (SIRP1α) (SEQ ID NO: 10) and theextracellular domain of CD40L, using the hinge-CH2-CH3 domain from ahuman IgG4 antibody sequence as a linker (this CD172a (SIRP1α)-Fc-CD40Lchimera is SEQ ID NO: 13).

In another embodiment, the chimeric protein of the present inventioncomprises the extracellular domain of PD-1 and the extracellular domainof TL1A (SEQ ID NO: 15).

Additional examples include a chimeric protein encoding theextracellular domain of BTLA, linked through an Fc to OX40L (SEQ ID NO:19).

Another example is a chimeric protein incorporating the extracellulardomain of TMIGD2 adjoined with an Fc linker sequence to theextracellular domain of human OX40L (SEQ ID NO: 22).

Another example is a chimeric protein incorporating the extracellulardomain of CD172a (SIRPα) adjoined with an Fc linker sequence to theextracellular domain of human OX40L (SEQ ID NO: 26).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of CSF1R (SEQ ID NO: 29).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of CSF1R (SEQ ID NO: 29) and an extracellulardomain of CD40L (SEQ ID NO: 12).

In embodiments, the chimeric protein of the present invention comprisesan extracellular domain of CSF1R (SEQ ID NO: 29) and an extracellulardomain of CD40L (SEQ ID NO: 12), using the hinge-CH2-CH3 domain from ahuman IgG4 antibody sequence as a linker (this CSF1R-Fc-CD40L chimera isSEQ ID NO: 30)

In embodiments, a chimeric protein can comprise an extracellular domainfrom a sequence identified herein combined with an extracellular domainfrom another sequence identified herein.

In embodiments, the present chimeric proteins may be variants describedherein, for instance, the present chimeric proteins may have a sequencehaving at least about 60%, or at least about 61%, or at least about 62%,or at least about 63%, or at least about 64%, or at least about 65%, orat least about 66%, or at least about 67%, or at least about 68%, or atleast about 69%, or at least about 70%, or at least about 71%, or atleast about 72%, or at least about 73%, or at least about 74%, or atleast about 75%, or at least about 76%, or at least about 77%, or atleast about 78%, or at least about 79%, or at least about 80%, or atleast about 81%, or at least about 82%, or at least about 83%, or atleast about 84%, or at least about 85%, or at least about 86%, or atleast about 87%, or at least about 88%, or at least about 89%, or atleast about 90%, or at least about 91%, or at least about 92%, or atleast about 93%, or at least about 94%, or at least about 95%, or atleast about 96%, or at least about 97%, or at least about 98%, or atleast about 99%) sequence identity with the amino acid sequence of thepresent chimeric proteins, e.g. one or more of SEQ IDs Nos 5, 5, 8, 13,16, 19, 22, 25, 26, 27, or 30.

In embodiments, the present chimeric proteins comprise an extracellulardomain of a human Type I transmembrane protein as recited in TABLE 1 ofPCT/US2016/054598, or a functional fragment thereof. In embodiments, thepresent chimeric proteins comprise an extracellular domain of a humanType II transmembrane protein as recited in TABLE 2 ofPCT/US2016/054598, or a functional fragment thereof. In embodiments, thepresent chimeric proteins comprise an extracellular domain of a Type Itransmembrane protein as recited in TABLE 1 of PCT/US2016/054598, or afunctional fragment thereof, and an extracellular domain of a Type IItransmembrane protein as recited in TABLE 2 of PCT/US2016/054598, or afunctional fragment thereof. The entire contents of PCT/US2016/054598are hereby incorporated by reference.

In embodiments, the chimeric protein may comprise an amino acid sequencehaving one or more amino acid mutations relative to any of the proteinsequences described herein. In embodiments, the one or more amino acidmutations may be independently selected from substitutions, insertions,deletions, and truncations.

In embodiments, the amino acid mutations are amino acid substitutions,and may include conservative and/or non-conservative substitutions.

“Conservative substitutions” may be made, for instance, on the basis ofsimilarity in polarity, charge, size, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the amino acid residuesinvolved. The 20 naturally occurring amino acids can be grouped into thefollowing six standard amino acid groups: (1) hydrophobic: Met, Ala,Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3)acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influencechain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe.

As used herein, “conservative substitutions” are defined as exchanges ofan amino acid by another amino acid listed within the same group of thesix standard amino acid groups shown above. For example, the exchange ofAsp by Glu retains one negative charge in the so modified polypeptide.In addition, glycine and proline may be substituted for one anotherbased on their ability to disrupt α-helices.

As used herein, “non-conservative substitutions” are defined asexchanges of an amino acid by another amino acid listed in a differentgroup of the six standard amino acid groups (1) to (6) shown above.

In embodiments, the substitutions may also include non-classical aminoacids (e.g., selenocysteine, pyrrolysine, N-formylmethionine β-alanine,GABA and δ-Aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers ofthe common amino acids, 2,4-diaminobutyric acid, α-amino isobutyricacid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu, ε-Ahx,6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionicacid, ornithine, norleucine, norvaline, hydroxyproline, sarcosme,citrulline, homocitrulline, cysteic acid, t-butylglycine,t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine,fluoro-amino acids, designer amino acids such as β methyl amino acids, Cα-methyl amino acids, N α-methyl amino acids, and amino acid analogs ingeneral).

Mutations may also be made to the nucleotide sequences of the chimericproteins by reference to the genetic code, including taking into accountcodon degeneracy.

In embodiments, the chimeric protein comprises a linker. In embodiments,the linker comprising at least one cysteine residue capable of forming adisulfide bond. As described elsewhere herein, such at least onecysteine residue capable of forming a disulfide bond is, without wishingto be bound by theory, responsible for maintain a proper multimericstate of the chimeric protein and allowing for efficient production.

In embodiments, there is provided a method of making a stable chimericprotein comprising adjoining a Type I and Type II transmembrane proteinextracellular domain with a linker comprising at least one cysteineresidue capable of forming a disulfide bond such that the resultantchimeric protein is properly folded and/or forms into a stablemultimeric state.

In embodiments, the linker may be derived from naturally-occurringmulti-domain proteins or are empirical linkers as described, forexample, in Chichili et al., (2013), Protein Sci. 22(2):153-167, Chen etal., (2013), Adv Drug Deliv Rev. 65(10):1357-1369, the entire contentsof which are hereby incorporated by reference. In embodiments, thelinker may be designed using linker designing databases and computerprograms such as those described in Chen et al., (2013), Adv Drug DelivRev. 65(10):1357-1369 and Crasto et. al., (2000), Protein Eng.13(5):309-312, the entire contents of which are hereby incorporated byreference.

In embodiments, the linker is a synthetic linker such as PEG.

In embodiments, the linker is a polypeptide. In embodiments, the linkeris less than about 500 amino acids long, about 450 amino acids long,about 400 amino acids long, about 350 amino acids long, about 300 aminoacids long, about 250 amino acids long, about 200 amino acids long,about 150 amino acids long, or about 100 amino acids long. For example,the linker may be less than about 100, about 95, about 90, about 85,about 80, about 75, about 70, about 65, about 60, about 55, about 50,about 45, about 40, about 35, about 30, about 25, about 20, about 19,about 18, about 17, about 16, about 15, about 14, about 13, about 12,about 11, about 10, about 9, about 8, about 7, about 6, about 5, about4, about 3, or about 2 amino acids long. In embodiments, the linker isflexible. In another embodiment, the linker is rigid.

In embodiments, the linker is substantially comprised of glycine andserine residues (e.g., about 30%, or about 40%, or about 50%, or about60%, or about 70%, or about 80%, or about 90%, or about 95%, or about97%, or about 98%, or about 99%, or about 100% glycines and serines).

In embodiments, the linker is a hinge region of an antibody (e.g., ofIgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., IgG1, IgG2, IgG3,and IgG4, and IgA1 and IgA2)). The hinge region, found in IgG, IgA, IgD,and IgE class antibodies, acts as a flexible spacer, allowing the Fabportion to move freely in space. In contrast to the constant regions,the hinge domains are structurally diverse, varying in both sequence andlength among immunoglobulin classes and subclasses. For example, thelength and flexibility of the hinge region varies among the IgGsubclasses. The hinge region of IgG1 encompasses amino acids 216-231and, because it is freely flexible, the Fab fragments can rotate abouttheir axes of symmetry and move within a sphere centered at the first oftwo inter-heavy chain disulfide bridges. IgG2 has a shorter hinge thanIgG1, with 12 amino acid residues and four disulfide bridges. The hingeregion of IgG2 lacks a glycine residue, is relatively short, andcontains a rigid poly-proline double helix, stabilized by extrainter-heavy chain disulfide bridges. These properties restrict theflexibility of the IgG2 molecule. IgG3 differs from the other subclassesby its unique extended hinge region (about four times as long as theIgG1 hinge), containing 62 amino acids (including 21 prolines and 11cysteines), forming an inflexible poly-proline double helix. In IgG3,the Fab fragments are relatively far away from the Fc fragment, givingthe molecule a greater flexibility. The elongated hinge in IgG3 is alsoresponsible for its higher molecular weight compared to the othersubclasses. The hinge region of IgG4 is shorter than that of IgG1 andits flexibility is intermediate between that of IgG1 and IgG2. Theflexibility of the hinge regions reportedly decreases in the orderIgG3>IgG1>IgG4>IgG2. In embodiments, the linker may be derived fromhuman IgG4 and contain one or more mutations to enhance dimerization(including S228P) or FcRn binding.

According to crystallographic studies, the immunoglobulin hinge regioncan be further subdivided functionally into three regions: the upperhinge region, the core region, and the lower hinge region. See Shin etal., 1992 Immunological Reviews 130:87. The upper hinge region includesamino acids from the carboxyl end of CH1 to the first residue in thehinge that restricts motion, generally the first cysteine residue thatforms an interchain disulfide bond between the two heavy chains. Thelength of the upper hinge region correlates with the segmentalflexibility of the antibody. The core hinge region contains theinter-heavy chain disulfide bridges, and the lower hinge region joinsthe amino terminal end of the C_(H2) domain and includes residues inC_(H2). Id. The core hinge region of wild-type human IgG1 contains thesequence Cys-Pro-Pro-Cys which, when dimerized by disulfide bondformation, results in a cyclic octapeptide believed to act as a pivot,thus conferring flexibility. In embodiments, the present linkercomprises, one, or two, or three of the upper hinge region, the coreregion, and the lower hinge region of any antibody (e.g., of IgG, IgA,IgD, and IgE, inclusive of subclasses (e.g., IgG1, IgG2, IgG3, and IgG4,and IgA1 and IgA2)). The hinge region may also contain one or moreglycosylation sites, which include a number of structurally distincttypes of sites for carbohydrate attachment. For example, IgA1 containsfive glycosylation sites within a 17-amino-acid segment of the hingeregion, conferring resistance of the hinge region polypeptide tointestinal proteases, considered an advantageous property for asecretory immunoglobulin. In embodiments, the linker of the presentinvention comprises one or more glycosylation sites.

In embodiments, the linker comprises an Fc domain of an antibody (e.g.,of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., IgG1, IgG2,IgG3, and IgG4, and IgA1 and IgA2)). In embodiments, the linkercomprises a hinge-CH2-CH3 Fc domain derived from a human IgG4 antibody.In embodiments, the linker comprises a hinge-CH2-CH3 Fc domain derivedfrom a human IgG1 antibody. In embodiments, the Fc domain exhibitsincreased affinity for and enhanced binding to the neonatal Fc receptor(FcRn). In embodiments, the Fc domain includes one or more mutationsthat increases the affinity and enhances binding to FcRn. Withoutwishing to be bound by theory, it is believed that increased affinityand enhanced binding to FcRn increases the in vivo half-life of thepresent chimeric proteins.

In embodiments, the Fc domain linker contains one or more amino acidsubstitutions at amino acid residue 250, 252, 254, 256, 308, 309, 311,416, 428, 433 or 434 (in accordance with Kabat numbering, as in as inKabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991) expressly incorporated herein by reference), or equivalentsthereof. In an embodiment, the amino acid substitution at amino acidresidue 250 is a substitution with glutamine. In an embodiment, theamino acid substitution at amino acid residue 252 is a substitution withtyrosine, phenylalanine, tryptophan or threonine. In an embodiment, theamino acid substitution at amino acid residue 254 is a substitution withthreonine. In an embodiment, the amino acid substitution at amino acidresidue 256 is a substitution with serine, arginine, glutamine, glutamicacid, aspartic acid, or threonine. In an embodiment, the amino acidsubstitution at amino acid residue 308 is a substitution with threonine.In an embodiment, the amino acid substitution at amino acid residue 309is a substitution with proline. In an embodiment, the amino acidsubstitution at amino acid residue 311 is a substitution with serine. Inan embodiment, the amino acid substitution at amino acid residue 385 isa substitution with arginine, aspartic acid, serine, threonine,histidine, lysine, alanine or glycine. In an embodiment, the amino acidsubstitution at amino acid residue 386 is a substitution with threonine,proline, aspartic acid, serine, lysine, arginine, isoleucine, ormethionine. In an embodiment, the amino acid substitution at amino acidresidue 387 is a substitution with arginine, proline, histidine, serine,threonine, or alanine. In an embodiment, the amino acid substitution atamino acid residue 389 is a substitution with proline, serine orasparagine. In an embodiment, the amino acid substitution at amino acidresidue 416 is a substitution with serine. In an embodiment, the aminoacid substitution at amino acid residue 428 is a substitution withleucine. In an embodiment, the amino acid substitution at amino acidresidue 433 is a substitution with arginine, serine, isoleucine,proline, or glutamine. In an embodiment, the amino acid substitution atamino acid residue 434 is a substitution with histidine, phenylalanine,or tyrosine.

In embodiments, the Fc domain linker (e.g., comprising an IgG constantregion) comprises one or more mutations such as substitutions at aminoacid residue 252, 254, 256, 433, 434, or 436 (in accordance with Kabatnumbering, as in as in Kabat, et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991) expressly incorporated hereinby reference). In an embodiment, the IgG constant region includes atriple M252Y/S254T/T256E mutation or YTE mutation. In anotherembodiment, the IgG constant region includes a triple H433K/N434F/Y436Hmutation or KFH mutation. In a further embodiment, the IgG constantregion includes an YTE and KFH mutation in combination.

In embodiments, the modified humanized antibodies of the inventioncomprise an IgG constant region that contains one or more mutations atamino acid residues 250, 253, 307, 310, 380, 416, 428, 433, 434, and435. Illustrative mutations include T250Q, M428L, T307A, E380A, 1253A,H310A, R416S, M428L, H433K, N434A, N434F, N434S, and H435A. In anembodiment, the IgG constant region comprises a M428L/N434S mutation orLS mutation. In another embodiment, the IgG constant region comprises aT250Q/M428L mutation or QL mutation. In another embodiment, the IgGconstant region comprises an N434A mutation. In another embodiment, theIgG constant region comprises a T307A/E380A/N434A mutation or MAmutation. In another embodiment, the IgG constant region comprises an1253A/H310A/H435A mutation or IHH mutation. In another embodiment, theIgG constant region comprises a H433K/N434F mutation. In anotherembodiment, the IgG constant region comprises a M252Y/S254T/T256E and aH433K/N434F mutation in combination.

Additional illustrative mutations in the IgG constant region aredescribed, for example, in Robbie, et al., Antimicrobial Agents andChemotherapy (2013), 57(12):6147-6153, Dall'Acqua et al., JBC (2006),281(33):23514-24, Dall'Acqua et al., Journal of Immunology (2002),169:5171-80, Ko et al., Nature (2014) 514:642-645, Grevys et al.,Journal of Immunology. (2015), 194(11):5497-508, and U.S. Pat. No.7,083,784, the entire contents of which are hereby incorporated byreference.

In embodiments, the linker comprises the amino acid sequence of SEQ IDNO: 45, or at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identitythereto. In embodiments, mutations are made to SEQ ID NO: 45 to increasestability and/or half-life. For instance, in embodiments, the linker hasthe amino acid sequence of SEQ ID NO: 46, or at least 90%, or 93%, or95%, or 97%, or 98%, or 99% identity thereto. In embodiments, the linkercomprises the amino acid sequence of SEQ ID NO: 47, or at least 90%, or93%, or 95%, or 97%, or 98%, or 99% identity thereto.

Without wishing to be bound by theory, including a linker comprising atleast a part of an Fc domain in a chimeric protein, helps avoidformation of insoluble and, likely, non-functional protein concatamersand/or aggregates. This is in part due to the presence of cysteines inthe Fc domain which are capable of forming disulfide bonds betweenchimeric proteins.

An illustrative Fc stabilizing mutant is S228P. Illustrative Fchalf-life extending mutants are T250Q, M428L, V308T, L309P, and Q311Sand the present linkers may comprise 1, or 2, or 3, or 4, or 5 of thesemutants.

Further, one or more joining linkers may be employed to connect an Fcdomain in a linker (e.g., one of SEQ ID NO: 45, SEQ ID NO: 46, or SEQ IDNO: 47 or at least 90%, or 93%, or 95%, or 97%, or 98%, or 99% identitythereto) and the extracellular domains. For example, any one of SEQ IDNO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQID NO: 53, or variants thereof may connect an extracellular domain asdescribed herein and a linker as described herein. Optionally, any oneof SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ IDNO: 52, SEQ ID NO: 53, or variants thereof are displaced between anextracellular domain as described herein and a linker as describedherein. Optionally, any one of SEQ ID NOs: 45 to 94, or variants thereofare located between an extracellular domain as described herein and anFc domain as described herein. In embodiments, a chimeric proteincomprises one joining linker preceding an Fc domain and a second joininglinker following the Fc domain; thus, a chimeric protein may comprisethe following structure:

-   -   ECD 1—Joining Linker 1—Fc Domain—Joining Linker 2—ECD 2.

In embodiments, the first and second joining linkers may be different orthey may be the same.

The amino acid sequences of illustrative linkers are provided in Table 1below:

TABLE 1 Illustrative linkers (Fc domain linkers and joining linkers)SEQ ID NO. Sequence 45APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSSWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK 46APEFLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTTPHSDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSSWQEGNVFSCSVLHEALHNHYTQKSLSLSLGK 47APEFLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSLGK 48 SKYGPPCPSCP 49 SKYGPPCPPCP 50SKYGPP 51 IEGRMD 52 GGGVPRDCG 53 IEGRMDGGGGAGGGG 54 GGGSGGGS 55GGGSGGGGSGGG 56 EGKSSGSGSESKST 57 GGSG 58 GGSGGGSGGGSG 59EAAAKEAAAKEAAAK 60 EAAAREAAAREAAAREAAAR 61 GGGGSGGGGSGGGGSAS 62GGGGAGGGG 63 GS or GGS or LE 64 GSGSGS 65 GSGSGSGSGS 66 GGGGSAS 67APAPAPAPAPAPAPAPAPAP 68 CPPC 69 GGGGS 70 GGGGSGGGGS 71 GGGGSGGGGSGGGGS72 GGGGSGGGGSGGGGSGGGGS 73 GGGGSGGGGSGGGGSGGGGSGGGGS 74GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 75 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 76GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS 77 GGSGGSGGGGSGGGGS 78 GGGGGGGG79 GGGGGG 80 EAAAK 81 EAAAKEAAAK 82 EAAAKEAAAKEAAAK 83 AEAAAKEAAAKA 84AEAAAKEAAAKEAAAKA 85 AEAAAKEAAAKEAAAKEAAAKA 86AEAAAKEAAAKEAAAKEAAAKEAAAKA 87AEAAAKEAAAKEAAAKEAAAKALEAEAAAKEAAAKEAAAKEAAAKA 88 PAPAP 89KESGSVSSEQLAQFRSLD 90 GSAGSAAGSGEF 91 GGGSE 92 GSESG 93 GSEGS 94GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS

Additional illustrative joining linkers include, but are not limited to,linkers having the sequence LE, GGGGS (SEQ ID NO: 69), (GGGGS)_(n)(n=1-4) (SEQ ID NO: 69-72), (Gly)₈ (SEQ ID NO: 78), (Gly)₆ (SEQ ID NO:79), (EAAAK)_(n) (n=1-3) (SEQ ID NO: 80-82), A(EAAAK)_(n)A (n=2-5) (SEQID NO: 83-86), AEAAAKEAAAKA (SEQ ID NO: 83), A(EAAAK)₄ALEA(EAAAK)₄A (SEQID NO: 87), PAPAP (SEQ ID NO: 88), KESGSVSSEQLAQFRSLD (SEQ ID NO: 89),EGKSSGSGSESKST (SEQ ID NO: 56), GSAGSAAGSGEF (SEQ ID NO: 90), and(XP)_(n), with X designating any amino acid, e.g., Ala, Lys, or Glu.

In embodiments, the joining linker is substantially comprised of glycineand serine residues (e.g., about 30%, or about 40%, or about 50%, orabout 60%, or about 70%, or about 80%, or about 90%, or about 95%, orabout 97%, or about 98%, or about 99%, or about 100%) glycines andserines). For example, in embodiments, the joining linker is(Gly₄Ser)_(n), where n is from about 1 to about 8, e.g., 1, 2, 3, 4, 5,6, 7, or 8 (SEQ ID NO: 69 to SEQ ID NO: 76, respectively). Inembodiments, the joining linker sequence is GGSGGSGGGGSGGGGS (SEQ ID NO:77). Additional illustrative joining linkers include, but are notlimited to, linkers having the sequence LE, (Gly)₈ (SEQ ID NO: 78),(Gly)₆ (SEQ ID NO: 79), (EAAAK)_(n) (n=1-3) (SEQ ID NO: 80-SEQ ID NO:82), A(EAAAK)_(n)A (n=2-5) (SEQ ID NO: 83-SEQ ID NO: 86),A(EAAAK)₄ALEA(EAAAK)₄A (SEQ ID NO: 43), PAPAP (SEQ ID NO: 44),KESGSVSSEQLAQFRSLD (SEQ ID NO: 45), GSAGSAAGSGEF (SEQ ID NO: 87), and(XP)_(n), with X designating any amino acid, e.g., Ala, Lys, or Glu. Inembodiments, the joining linker is GGS.

In embodiments, the joining linker is one or more of GGGSE (SEQ ID NO:91), GSESG (SEQ ID NO: 92), GSEGS (SEQ ID NO: 93),GEGGSGEGSSGEGSSSEGGGSEGGGSEGGGSEGGS (SEQ ID NO: 94), and a joininglinker of randomly placed G, S, and E every 4 amino acid intervals.

In embodiments, a chimeric protein comprises a modular linker as shownin FIG. 39.

In embodiments, the linker may be flexible, including without limitationhighly flexible. In embodiments, the linker may be rigid, includingwithout limitation a rigid alpha helix.

In embodiments, the linker may be functional. For example, withoutlimitation, the linker may function to improve the folding and/orstability, improve the expression, improve the pharmacokinetics, and/orimprove the bioactivity of the present chimeric protein. In anotherexample, the linker may function to target the chimeric protein to aparticular cell type or location.

In embodiments, the present chimeric proteins are capable of, and can beused in methods comprising, promoting immune activation (e.g., againsttumors). In embodiments, the present chimeric proteins are capable of,and can be used in methods comprising, suppressing immune inhibition(e.g., that allows tumors to survive). In embodiments, the presentchimeric proteins provide improved immune activation and/or improvedsuppression of immune inhibition due to the proximity of signaling thatis provided by the chimeric nature of the constructs.

In embodiments, the present chimeric proteins are capable of, or can beused in methods comprising, modulating the amplitude of an immuneresponse, e.g., modulating the level of effector output. In embodiments,e.g., when used for the treatment of cancer, the present chimericproteins alter the extent of immune stimulation as compared to immuneinhibition to increase the amplitude of a T cell response, including,without limitation, stimulating increased levels of cytokine production,proliferation or target killing potential.

In embodiments the present chimeric proteins, in embodiments are capableof, or find use in methods involving, masking an inhibitory ligand onthe surface of a tumor cell and replacing that immune inhibitory ligandwith an immune stimulatory ligand. Accordingly, the present chimericproteins, in embodiments are capable of, or find use in methodsinvolving, reducing or eliminating an inhibitory immune signal and/orincreasing or activating an immune stimulatory signal. For example, atumor cell bearing an inhibitory signal (and thus evading an immuneresponse) may be substituted for a positive signal binding on a T cellthat can then attack a tumor cell. Accordingly, in embodiments, aninhibitory immune signal is masked by the present constructs and astimulatory immune signal is activated. Such beneficial properties areenhanced by the single construct approach of the present chimericproteins. For instance, the signal replacement can be effected nearlysimultaneously and the signal replacement is tailored to be local at asite of clinical importance (e.g., the tumor microenvironment). Furtherembodiments apply the same principle to other chimeric proteinconstructs, such as, for example, (i) the extracellular domain of PD-1and (ii) extracellular domain of GITRL; (i) the extracellular domain ofBTLA and (ii) extracellular domain of OX40L; (i) the extracellulardomain of TIGIT and (ii) extracellular domain of OX40L; (i) theextracellular domain of TIM3 and (ii) extracellular domain of OX40L; and(i) the extracellular domain of CD172a (SIRP1α) and (ii) extracellulardomain of CD40L; and (i) the extracellular domain of CD115 and (ii)extracellular domain of CD40L; and (i) the extracellular domain of TIM3and (ii) extracellular domain of OX40L; and (i) the extracellular domainof TIGIT and (ii) extracellular domain of OX40L; among others.

In embodiments, the present chimeric proteins are capable of, or finduse in methods comprising, stimulating or enhancing the binding ofimmune stimulatory receptor/ligand pairs. Illustrative T cellcostimulatory receptors and their ligands include OX-40:OX40-L,CD27:CD70, CD30:CD30-L, CD40:CD40-L; CD137:CD137-L, HVEM:LIGHT,GITR:GITR-L, TNFRSF25:TL1A, DR5:TRAIL, and BTLA:HVEM. In embodiments,the present chimeric proteins are capable of, or find use in methodscomprising, inhibiting or reducing the binding of immune inhibitoryreceptor/ligand pairs. Illustrative T cell coinhibitory receptors andtheir ligands include, for example, CTLA-4:CD80/CD86, PD-1:PD-L1/PD-L2,BTLA:HVEM, TIM-3:galectin-9/phosphatidylserine, TIGIT/CD155 or CD112,VISTA/VSIG8, CD172a (SIRPα)/CD47, B7H3R/B7H3, B7H4R/B7H4, CD244/CD48,TMIGD2/HHLA2, among others.

In embodiments, the present chimeric protein blocks, reduces and/orinhibits PD-1 and PD-L1 or PD-L2 and/or the binding of PD-1 with PD-L1or PD-L2. In embodiments, the present chimeric protein blocks, reducesand/or inhibits the activity of CTLA-4 and/or the binding of CTLA-4 withone or more of AP2M1, CD80, CD86, SHP-2, and PPP2R5A. In embodiments,the present chimeric protein increases and/or stimulates GITR and/or thebinding of GITR with one or more of GITR ligand. In embodiments, thepresent chimeric protein increases and/or stimulates OX40 and/or thebinding of OX40 with one or more of OX40 ligand.

In embodiments, the present chimeric proteins are capable of, or finduse in methods involving, enhancing, restoring, promoting and/orstimulating immune modulation. In embodiments, the present chimericproteins described herein, restore, promote and/or stimulate theactivity or activation of one or more immune cells against tumor cellsincluding, but not limited to: T cells, cytotoxic T lymphocytes, Thelper cells, natural killer (NK) cells, natural killer T (NKT) cells,anti-tumor macrophages (e.g., M1 macrophages), B cells, and dendriticcells. In embodiments, the present chimeric proteins enhance, restore,promote and/or stimulate the activity and/or activation of T cells,including, by way of a non-limiting example, activating and/orstimulating one or more T− cell intrinsic signals, including apro-survival signal; an autocrine or paracrine growth signal; a p38MAPK−, ERK−, STAT-, JAK-, AKT- or PI3K-mediated signal; ananti-apoptotic signal; and/or a signal promoting and/or necessary forone or more of: proinflammatory cytokine production or T cell migrationor T cell tumor infiltration.

In embodiments, the present chimeric proteins are capable of, or finduse in methods involving, causing an increase of one or more of T cells(including without limitation cytotoxic T lymphocytes, T helper cells,natural killer T (NKT) cells), B cells, natural killer (NK) cells,natural killer T (NKT) cells, dendritic cells, monocytes, andmacrophages (e.g., one or more of M1 and M2) into a tumor or the tumormicroenvironment. In embodiments, the present chimeric proteins arecapable of, or find use in methods involving, inhibiting and/or causinga decrease in recruitment of immunosuppressive cells (e.g.,myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs),tumor associated neutrophils (TANs), M2 macrophages, and tumorassociated macrophages (TAMs)) to the tumor and/or tumormicroenvironment (TME). In embodiments, the present therapies may alterthe ratio of M1 versus M2 macrophages in the tumor site and/or TME tofavor M1 macrophages.

In embodiments, the present chimeric proteins are capable of, and can beused in methods comprising, inhibiting and/or reducing T cellinactivation and/or immune tolerance to a tumor, comprisingadministering an effective amount of a chimeric protein described hereinto a subject. In embodiments, the present chimeric proteins are able toincrease the serum levels of various cytokines including, but notlimited to, one or more of IFNγ, TNFα, IL-2, IL-4, IL-5, IL-6, IL-9,IL-10, IL-13, IL-17A, IL-17F, and IL-22. In embodiments, the presentchimeric proteins are capable of enhancing IL-2, IL-4, IL-5, IL-10,IL-13, IL-17A, IL-22, TNFα or IFNγ in the serum of a treated subject. Inembodiments, administration of the present chimeric protein is capableof enhancing TNFα secretion. In a specific embodiment, administration ofthe present chimeric protein is capable of enhancing superantigenmediated TNFα secretion by leukocytes. Detection of such a cytokineresponse may provide a method to determine the optimal dosing regimenfor the indicated chimeric protein.

In embodiments, the present chimeric proteins inhibit, block and/orreduce cell death of an anti-tumor CD8+ and/or CD4+ T cell; orstimulate, induce, and/or increase cell death of a pro-tumor T cell. Tcell exhaustion is a state of T cell dysfunction characterized byprogressive loss of proliferative and effector functions, culminating inclonal deletion. Accordingly, a pro-tumor T cell refers to a state of Tcell dysfunction that arises during many chronic infections and cancer.This dysfunction is defined by poor proliferative and/or effectorfunctions, sustained expression of inhibitory receptors and atranscriptional state distinct from that of functional effector ormemory T cells. Exhaustion prevents optimal control of infection andtumors. In addition, an anti-tumor CD8+ and/or CD4+ T cell refers to Tcells that can mount an immune response to a tumor. Illustrativepro-tumor T cells include, but are not limited to, Tregs, CD4+ and/orCD8+ T cells expressing one or more checkpoint inhibitory receptors, Th2cells and Th17 cells. Checkpoint inhibitory receptors refers toreceptors (e.g., CTLA-4, B7-H3, B7-H4, TIM-3) expressed on immune cellsthat prevent or inhibit uncontrolled immune responses.

In embodiments, the present chimeric proteins are capable of, and can beused in methods comprising, increasing a ratio of effector T cells toregulatory T cells. Illustrative effector T cells include ICOS⁺ effectorT cells; cytotoxic T cells (e.g., αβ TCR, CD3⁺, CD8⁺, CD45RO⁺); CD4⁺effector T cells (e.g., αβ TCR, CD3⁺, CD4⁺, CCR7⁺, CD62Lhi, IL⁻7R/CD127⁺); CD8⁺ effector T cells (e.g., αβ TCR, CD3⁺, CD8⁺, CCR7⁺,CD62Lhi, IL⁻7 R/CD127⁺); effector memory T cells (e.g., CD62Llow, CD44⁺,TCR, CD3⁺, IL⁻7 R/CD127⁺, IL-15R⁺, CCR7low); central memory T cells(e.g., CCR7⁺, CD62L⁺, CD27⁺; or CCR7hi, CD44⁺, CD62Lhi, TCR, CD3⁺,IL-7R/CD127⁺, IL-15R⁺); CD62L⁺ effector T cells; CD8⁺ effector memory Tcells (TEM) including early effector memory T cells (CD27⁺ CD62L⁻) andlate effector memory T cells (CD27⁻ CD62L⁻) (TemE and TemL,respectively); CD127(⁺)CD25(low/−) effector T cells; CD127(⁻)CD25(⁻)effector T cells; CD8′ stem cell memory effector cells (TSCM) (e.g.,CD44(low)CD62L(high)CD122(high)sca(⁺)); TH1 effector T-cells (e.g.,CXCR3⁺, CXCR6⁺ and CCR5⁺; or αβ TCR, CD3⁺, CD4⁺, IL-12R⁺, IFNγR⁺,CXCR3⁺), TH2 effector T cells (e.g., CCR3⁺, CCR4⁺ and CCR8⁺; or αβ TCR,CD3⁺, CD4⁺, IL-4R⁺, IL-33R⁺, CCR4⁺, IL-17RB⁺, CRTH2⁺); TH9 effector Tcells (e.g., αβ TCR, CD3⁺, CD4′); TH17 effector T cells (e.g., αβ TCR,CD3⁺, CD4⁺, IL-23R⁺, CCR6⁺, IL-1R⁺); CD4⁺CD45RO⁺CCR7⁺ effector T cells,CD4⁺CD45RO⁺CCR7(⁻) effector T cells; and effector T cells secretingIL-2, IL-4 and/or IFN-γ. Illustrative regulatory T cells include ICOS⁺regulatory T cells, CD4⁺CD25⁺FOXP3⁺ regulatory T cells, CD4⁺CD25⁺regulatory T cells, CD4⁺CD25⁻ regulatory T cells, CD4⁺CD25_(high)regulatory T cells, TIM-3⁺PD-1⁺ regulatory T cells, lymphocyteactivation gene-3 (LAG-3)⁺ regulatory T cells, CTLA-4/CD152⁺ regulatoryT cells, neuropilin-1 (Nrp-1)⁺ regulatory T cells, CCR4⁺CCR8⁺ regulatoryT cells, CD62L (L-selectin)′ regulatory T cells, CD45RBlow regulatory Tcells, CD127low regulatory T cells, LRRC32/GARP⁺ regulatory T cells,CD39⁺ regulatory T cells, GITR⁺ regulatory T cells, LAP′ regulatory Tcells, 1B11⁺ regulatory T cells, BTLA⁺ regulatory T cells, type 1regulatory T cells (Tr1 cells), T helper type 3 (Th3) cells, regulatorycell of natural killer T cell phenotype (NKTregs), CD8′ regulatory Tcells, CD8⁺CD28⁻ regulatory T cells and/or regulatory T-cells secretingIL-10, IL-35, TGF-β, TNF-α, Galectin-1, IFN-γ and/or MCP1.

In embodiments, the chimeric protein generates a memory response whichmay, e.g., be capable of preventing relapse or protecting the animalfrom a recurrence and/or preventing, or reducing the likelihood of,metastasis. Thus, an animal treated with the chimeric protein is laterable to attack tumor cells and/or prevent development of tumors whenexposed to the relevant antigen after an initial treatment with thechimeric protein. Accordingly, a chimeric protein of the presentinvention stimulates both active tumor destruction and also immunerecognition of tumor antigens, which are essential in programming amemory response capable of preventing relapse.

In embodiments, the present chimeric proteins are capable of, and can beused in methods comprising, transiently stimulating effector T cells forno longer than about 12 hours, about 24 hours, about 48 hours, about 72hours or about 96 hours or about 1 week or about 2 weeks. Inembodiments, the present chimeric proteins are capable of, and can beused in methods comprising, transiently depleting or inhibitingregulatory T cells for no longer than about 12 hours, about 24 hours,about 48 hours, about 72 hours or about 96 hours or about 1 week orabout 2 weeks. In embodiments, the transient stimulation of effector Tcells and/or transient depletion or inhibition of regulatory T cellsoccurs substantially in a patient's bloodstream or in a particulartissue/location including lymphoid tissues such as for example, the bonemarrow, lymph-node, spleen, thymus, mucosa-associated lymphoid tissue(MALT), non-lymphoid tissues, or in the tumor microenvironment.

In embodiments, the present chimeric proteins provide advantagesincluding, without limitation, ease of use and ease of production. Thisis because two distinct immunotherapy agents are combined into a singleproduct which allows for a single manufacturing process instead of twoindependent manufacturing processes. In addition, administration of asingle agent instead of two separate agents allows for easieradministration and greater patient compliance. Further, in contrast to,for example, monoclonal antibodies, which are large multimeric proteinscontaining numerous disulfide bonds and post-translational modificationssuch as glycosylation, the present chimeric proteins are easier and morecost effective to manufacture.

In embodiments, the present chimeric protein is producible in amammalian host cell as a secretable and fully functional singlepolypeptide chain.

In embodiments, the present chimeric protein unexpectedly providesbinding of the extracellular domain components to their respectivebinding partners with slow off rates (K_(d) or K_(off)). In embodiments,this provides an unexpectedly long interaction of the receptor to ligandand vice versa. Such an effect allows for a sustained negative signalmasking effect. Further, in embodiments, this delivers a longer positivesignal effect, e.g., to allow an effector cell to be adequatelystimulated for an anti-tumor effect. For example, the present chimericprotein, e.g., via the long off rate binding allows sufficient signaltransmission to provide T cell proliferation and allow for anti-tumorattack. By way of further example, the present chimeric protein, e.g.,via the long off rate binding allows sufficient signal transmission toprovide release of stimulatory signals, such as, for example, cytokines.

The stable synapse of cells promoted by the present agents (e.g., atumor cell bearing negative signals and a T cell which could attack thetumor) provides spatial orientation to favor tumor reduction—such aspositioning the T cells to attack tumor cells and/or stericallypreventing the tumor cell from delivering negative signals, includingnegative signals beyond those masked by the chimeric protein of theinvention.

In embodiments, this provides longer on-target (e.g., intra-tumoral)half-life (t_(1/2)) as compared to serum t_(1/2) of the chimericproteins. Such properties could have the combined advantage of reducingoff-target toxicities which may be associated with systemic distributionof the chimeric proteins.

Further, in embodiments, the present chimeric proteins providesynergistic therapeutic effects as it allows for improved site-specificinterplay of two immunotherapy agents.

In embodiments, the present chimeric proteins provide the potential forreducing off-site and/or systemic toxicity.

In embodiments, the present chimeric proteins provide reducedside-effects, e.g., GI complications, relative to currentimmunotherapies, e.g., antibodies directed to checkpoint molecules asdescribed herein. Illustrative GI complications include abdominal pain,appetite loss, autoimmune effects, constipation, cramping, dehydration,diarrhea, eating problems, fatigue, flatulence, fluid in the abdomen orascites, gastrointestinal (GI) dysbiosis, GI mucositis, inflammatorybowel disease, irritable bowel syndrome (IBS-D and IBS-C), nausea, pain,stool or urine changes, ulcerative colitis, vomiting, weight gain fromretaining fluid, and/or weakness

Diseases; Methods of Treatment, and Patient Selections

In embodiments, the present invention pertains to cancers and/or tumors;for example, the treatment or prevention of cancers and/or tumors. Asdescribed elsewhere herein, the treatment of cancer may involve inembodiments, modulating the immune system with the present chimericproteins to favor immune stimulation over immune inhibition.

Cancers or tumors refer to an uncontrolled growth of cells and/orabnormal increased cell survival and/or inhibition of apoptosis whichinterferes with the normal functioning of the bodily organs and systems.Included are benign and malignant cancers, polyps, hyperplasia, as wellas dormant tumors or micrometastases. Also, included are cells havingabnormal proliferation that is not impeded by the immune system (e.g.,virus infected cells). The cancer may be a primary cancer or ametastatic cancer. The primary cancer may be an area of cancer cells atan originating site that becomes clinically detectable, and may be aprimary tumor. In contrast, the metastatic cancer may be the spread of adisease from one organ or part to another non-adjacent organ or part.The metastatic cancer may be caused by a cancer cell that acquires theability to penetrate and infiltrate surrounding normal tissues in alocal area, forming a new tumor, which may be a local metastasis. Thecancer may also be caused by a cancer cell that acquires the ability topenetrate the walls of lymphatic and/or blood vessels, after which thecancer cell is able to circulate through the bloodstream (thereby beinga circulating tumor cell) to other sites and tissues in the body. Thecancer may be due to a process such as lymphatic or hematogenous spread.The cancer may also be caused by a tumor cell that comes to rest atanother site, re-penetrates through the vessel or walls, continues tomultiply, and eventually forms another clinically detectable tumor. Thecancer may be this new tumor, which may be a metastatic (or secondary)tumor.

The cancer may be caused by tumor cells that have metastasized, whichmay be a secondary or metastatic tumor. The cells of the tumor may belike those in the original tumor. As an example, if a breast cancer orcolon cancer metastasizes to the liver, the secondary tumor, whilepresent in the liver, is made up of abnormal breast or colon cells, notof abnormal liver cells. The tumor in the liver may thus be a metastaticbreast cancer or a metastatic colon cancer, not liver cancer.

The cancer may have an origin from any tissue. The cancer may originatefrom melanoma, colon, breast, or prostate, and thus may be made up ofcells that were originally skin, colon, breast, or prostate,respectively. The cancer may also be a hematological malignancy, whichmay be leukemia or lymphoma. The cancer may invade a tissue such asliver, lung, bladder, or intestinal.

Representative cancers and/or tumors of the present invention include,but are not limited to, a basal cell carcinoma, biliary tract cancer;bladder cancer; bone cancer; brain and central nervous system cancer;breast cancer; cancer of the peritoneum; cervical cancer;choriocarcinoma; colon and rectum cancer; connective tissue cancer;cancer of the digestive system; endometrial cancer; esophageal cancer;eye cancer; cancer of the head and neck; gastric cancer (includinggastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma;intra-epithelial neoplasm; kidney or renal cancer; larynx cancer;leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer,non-small cell lung cancer, adenocarcinoma of the lung, and squamouscarcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavitycancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreaticcancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectalcancer; cancer of the respiratory system; salivary gland carcinoma;sarcoma; skin cancer; squamous cell cancer; stomach cancer; testicularcancer; thyroid cancer; uterine or endometrial cancer; cancer of theurinary system; vulval cancer; lymphoma including Hodgkin's andnon-Hodgkin's lymphoma, as well as B-cell lymphoma (including lowgrade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL)NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL;high grade immunoblastic NHL; high grade lymphoblastic NHL; high gradesmall non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chroniclymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); Hairycell leukemia; chronic myeloblastic leukemia; as well as othercarcinomas and sarcomas; and post-transplant lymphoproliferativedisorder (PTLD), as well as abnormal vascular proliferation associatedwith phakomatoses, edema (such as that associated with brain tumors),and Meigs' syndrome.

In embodiments, the chimeric protein is used to treat a subject that hasa treatment-refractory cancer. In embodiments, the chimeric protein isused to treat a subject that is refractory to one or moreimmune-modulating agents. For example, in embodiments, the chimericprotein is used to treat a subject that presents no response totreatment, or even progress, after 12 weeks or so of treatment. Forinstance, in embodiments, the subject is refractory to α PD-1 and/orPD-L1 and/or PD-L2 agent, including, for example, nivolumab(ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB),pembrolizumab (KEYTRUDA, MERCK), pidilizumab (CT-011, CURE TECH),MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), Ibrutinib(PHARMACYCLICS/ABBVIE), atezolizumab (TECENTRIQ, GENENTECH), and/orMPDL328OA (ROCHE)-refractory patients. For instance, in embodiments, thesubject is refractory to an anti-CTLA-4 agent, e.g., ipilimumab(YERVOY)-refractory patients (e.g., melanoma patients). Accordingly, inembodiments the present invention provides methods of cancer treatmentthat rescue patients that are non-responsive to various therapies,including monotherapy of one or more immune-modulating agents.

In embodiments, the present invention provides chimeric proteins whichtarget a cell or tissue within the tumor microenvironment. Inembodiments, the cell or tissue within the tumor microenvironmentexpresses one or more targets or binding partners of the chimericprotein. The tumor microenvironment refers to the cellular milieu,including cells, secreted proteins, physiological small molecules, andblood vessels in which the tumor exists. In embodiments, the cells ortissue within the tumor microenvironment are one or more of: tumorvasculature; tumor-infiltrating lymphocytes; fibroblast reticular cells;endothelial progenitor cells (EPC); cancer-associated fibroblasts;pericytes; other stromal cells; components of the extracellular matrix(ECM); dendritic cells; antigen presenting cells; T-cells; regulatory Tcells; macrophages; neutrophils; and other immune cells located proximalto a tumor. In embodiments, the present chimeric protein targets acancer cell. In embodiments, the cancer cell expresses one or more oftargets or binding partners of the chimeric protein.

In an illustrative embodiment, the chimeric protein of the invention maytarget a cell (e.g., cancer cell or immune cell) that expresses PD-L1and/or PD-L2. In an illustrative embodiment, the chimeric protein maytarget a cell (e.g., cancer cell or immune cell) that expresses OX-40.In an illustrative embodiment, the chimeric protein may target a cell(e.g., cancer cell or immune cell) that expresses GITR. In anillustrative embodiment, the chimeric protein may target a cell (e.g.,cancer cell or immune cell) that expresses 4-1BB. In an illustrativeembodiment, the chimeric protein may target a cell (e.g., cancer cell orimmune cell) that expresses CD40. In an illustrative embodiment, thechimeric protein may target a cell (e.g., cancer cell or immune cell)that expresses VISTA. In an illustrative embodiment, the chimericprotein may target a cell (e.g., cancer cell or immune cell) thatexpresses CSF1. In an illustrative embodiment, the chimeric protein maytarget a cell (e.g., cancer cell or immune cell) that expresses IL-34.In an illustrative embodiment, the chimeric protein may target a cell(e.g., cancer cell or immune cell) that expresses CD47. In anillustrative embodiment, the chimeric protein may target a cell (e.g.,cancer cell, stromal cell or immune cell) that expresses galectin-9and/or phosphatidyserine.

In embodiments, the present methods provide treatment with the chimericprotein in a patient who is refractory to an additional agent, such“additional agents” being described elsewhere herein, inclusive, withoutlimitation, of the various chemotherapeutic agents described herein.

In embodiments, the chimeric proteins are used to treat, control orprevent one or more inflammatory diseases or conditions. Non-limitingexamples of inflammatory diseases include acne vulgaris, acuteinflammation, allergic rhinitis, asthma, atherosclerosis, atopicdermatitis, autoimmune disease, autoinflammatory diseases, autosomalrecessive spastic ataxia, bronchiectasis, celiac disease, chroniccholecystitis, chronic inflammation, chronic prostatitis, colitis,diverticulitis, familial eosinophilia (fe), glomerulonephritis, glycerolkinase deficiency, hidradenitis suppurativa, hypersensitivities,inflammation, inflammatory bowel diseases, inflammatory pelvic disease,interstitial cystitis, laryngeal inflammatory disease, Leigh syndrome,lichen planus, mast cell activation syndrome, mastocytosis, ocularinflammatory disease, otitis, pain, pelvic inflammatory disease,reperfusion injury, respiratory disease, restenosis, rheumatic fever,rheumatoid arthritis, rhinitis, sarcoidosis, septic shock, silicosis andother pneumoconiosis, transplant rejection, tuberculosis, andvasculitis.

In embodiments, the inflammatory disease is an autoimmune disease orcondition, such as multiple sclerosis, diabetes mellitus, lupus, celiacdisease, Crohn's disease, ulcerative colitis, Guillain-Barre syndrome,scleroderms, Goodpasture's syndrome, Wegener's granulomatosis,autoimmune epilepsy, Rasmussen's encephalitis, Primary biliarysclerosis, Sclerosing cholangitis, Autoimmune hepatitis, Addison'sdisease, Hashimoto's thyroiditis, Fibromyalgia, Menier's syndrome;transplantation rejection (e.g., prevention of allograft rejection)pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus,dermatomyositis, Sjogren's syndrome, lupus erythematosus, multiplesclerosis, myasthenia gravis, Reiter's syndrome, Grave's disease, andother autoimmune diseases.

In some aspects, the present chimeric agents are used to eliminateintracellular pathogens. In some aspects, the present chimeric agentsare used to treat one or more infections. In embodiments, the presentchimeric proteins are used in methods of treating viral infections(including, for example, HIV and HCV), parasitic infections (including,for example, malaria), and bacterial infections. In embodiments, theinfections induce immunosuppression. For example, HIV infections oftenresult in immunosuppression in the infected subjects. Accordingly, asdescribed elsewhere herein, the treatment of such infections mayinvolve, in embodiments, modulating the immune system with the presentchimeric proteins to favor immune stimulation over immune inhibition.Alternatively, the present invention provides methods for treatinginfections that induce immunoactivation. For example, intestinalhelminth infections have been associated with chronic immune activation.In these embodiments, the treatment of such infections may involvemodulating the immune system with the present chimeric proteins to favorimmune inhibition over immune stimulation.

In embodiments, the present invention provides methods of treating viralinfections including, without limitation, acute or chronic viralinfections, for example, of the respiratory tract, of papilloma virusinfections, of herpes simplex virus (HSV) infection, of humanimmunodeficiency virus (HIV) infection, and of viral infection ofinternal organs such as infection with hepatitis viruses. Inembodiments, the viral infection is caused by a virus of familyFlaviviridae. In embodiments, the virus of family Flaviviridae isselected from Yellow Fever Virus, West Nile virus, Dengue virus,Japanese Encephalitis Virus, St. Louis Encephalitis Virus, and HepatitisC Virus. In embodiments, the viral infection is caused by a virus offamily Picornaviridae, e.g., poliovirus, rhinovirus, coxsackievirus. Inembodiments, the viral infection is caused by a member ofOrthomyxoviridae, e.g., an influenza virus. In embodiments, the viralinfection is caused by a member of Retroviridae, e.g., a lentivirus. Inembodiments, the viral infection is caused by a member ofParamyxoviridae, e.g., respiratory syncytial virus, a humanparainfluenza virus, rubulavirus (e.g., mumps virus), measles virus, andhuman metapneumovirus. In embodiments, the viral infection is caused bya member of Bunyaviridae, e.g., hantavirus. In embodiments, the viralinfection is caused by a member of Reoviridae, e.g., a rotavirus.

In embodiments, the present invention provides methods of treatingparasitic infections such as protozoan or helminths infections. Inembodiments, the parasitic infection is by a protozoan parasite. Inembodiments, the oritiziab parasite is selected from intestinalprotozoa, tissue protozoa, or blood protozoa. Illustrative protozoanparasites include, but are not limited to, Entamoeba, Giardia lamblia,Cryptosporidium muris, Trypanosomatida gambiense, Trypanosomatidarhodesiense, Trypanosomatida crusi, Leishmania mexicana, Leishmaniabraziliensis, Leishmania tropica, Leishmania donovani, Toxoplasmagondii, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae,Plasmodium falciparum, Trichomonas vaginalis, and Histomonasmeleagridis. In embodiments, the parasitic infection is by a helminthicparasite such as nematodes (e.g., Adenophorea). In embodiments, theparasite is selected from Secementea (e.g., Trichuris trichiura, Ascarislumbricoides, Enterobius vermicularis, Ancylostoma duodenale, Necatoramericanus, Strongyloides stercoralis, Wuchereria bancrofti, Dracunculusmedinensis). In embodiments, the parasite is selected from trematodes(e.g., blood flukes, liver flukes, intestinal flukes, and lung flukes).In embodiments, the parasite is selected from: Schistosoma mansoni,Schistosoma haematobium, Schistosoma japonicum, Fasciola hepatica,Fasciola gigantica, Heterophyes heterophyes, Paragonimus westermani. Inembodiments, the parasite is selected from cestodes (e.g., Taeniasolium, Taenia saginata, Hymenolepis nana, Echinococcus granulosus).

In embodiments, the present invention provides methods of treatingbacterial infections. In embodiments, the bacterial infection is bygram-positive bacteria, gram-negative bacteria, aerobic and/or anaerobicbacteria. In embodiments, the bacteria is selected from, but not limitedto, Staphylococcus, Lactobacillus, Streptococcus, Sarcina, Escherichia,Enterobacter, Klebsiella, Pseudomonas, Acinetobacter, Mycobacterium,Proteus, Campylobacter, Citrobacter, Nisseria, Baccillus, Bacteroides,Peptococcus, Clostridium, Salmonella, Shigella, Serratia, Haemophilus,Brucella and other organisms. In embodiments, the bacteria is selectedfrom, but not limited to, Pseudomonas aeruginosa, Pseudomonasfluorescens, Pseudomonas acidovorans, Pseudomonas alcaligenes,Pseudomonas putida, Stenotrophomonas maltophilia, Burkholderia cepacia,Aeromonas hydrophilia, Escherichia coli, Citrobacter freundii,Salmonella typhimurium, Salmonella typhi, Salmonella paratyphi,Salmonella enteritidis, Shigella dysenteriae, Shigella flexneri,Shigella sonnei, Enterobacter cloacae, Enterobacter aerogenes,Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens,Francisella tularensis, Morganella morganii, Proteus mirabilis, Proteusvulgaris, Providencia alcalifaciens, Providencia rettgeri, Providenciastuartii, Acinetobacter baumannii, Acinetobacter calcoaceticus,Acinetobacter haemolyticus, Yersinia enterocolitica, Yersinia pestis,Yersinia pseudotuberculosis, Yersinia intermedia, Bordetella pertussis,Bordetella parapertussis, Bordetella bronchiseptica, Haemophilusinfluenzae, Haemophilus parainfluenzae, Haemophilus haemolyticus,Haemophilus parahaemolyticus, Haemophilus ducreyi, Pasteurellamultocida, Pasteurella haemolytica, Branhamella catarrhalis,Helicobacter pylori, Campylobacter fetus, Campylobacter jejuni,Campylobacter coli, Borrelia burgdorferi, Vibrio cholerae, Vibrioparahaemolyticus, Legionella pneumophila, Listeria monocytogenes,Neisseria gonorrhoeae, Neisseria meningitidis, Kingella, Moraxella,Gardnerella vaginalis, Bacteroides fragilis, Bacteroides distasonis,Bacteroides 3452A homology group, Bacteroides vulgatus, Bacteroidesovalus, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroideseggerthii, Bacteroides splanchnicus, Clostridium difficile,Mycobacterium tuberculosis, Mycobacterium avium, Mycobacteriumintracellulare, Mycobacterium leprae, Corynebacterium diphtheriae,Corynebacterium ulcerans, Streptococcus pneumoniae, Streptococcusagalactiae, Streptococcus pyogenes, Enterococcus faecalis, Enterococcusfaecium, Staphylococcus aureus, Staphylococcus epidermidis,Staphylococcus saprophyticus, Staphylococcus intermedius, Staphylococcushyicus subsp. hyicus, Staphylococcus haemolyticus, Staphylococcushominis, or Staphylococcus saccharolyticus.

In some aspects, the present chimeric agents are used to treat one ormore autoimmune diseases or disorders. In embodiments, the treatment ofan autoimmune disease or disorder may involve modulating the immunesystem with the present chimeric proteins to favor immune inhibitionover immune stimulation. Illustrative autoimmune diseases or disorderstreatable with the present chimeric proteins include those in which thebody's own antigens become targets for an immune response, such as, forexample, rheumatoid arthritis, systemic lupus erythematosus, diabetesmellitus, ankylosing spondylitis, Sjögren's syndrome, inflammatory boweldiseases (e.g., colitis ulcerosa, Crohn's disease), multiple sclerosis,sarcoidosis, psoriasis, Grave's disease, Hashimoto's thyroiditis,psoriasis, hypersensitivity reactions (e.g., allergies, hay fever,asthma, and acute edema cause Type I hypersensitivity reactions), andvasculitis.

In still another other aspect, the present invention is directed towardmethods of treating and preventing T cell-mediated diseases anddisorders, such as, but not limited to diseases or disorders describedelsewhere herein and inflammatory disease or disorder, graft-versus-hostdisease (GVHD), transplant rejection, and T cell proliferative disorder.Specific examples of Type I ECD domains with utility in this method ofuse include but are not limited to: TNFRSF1b, BTNL2, PD-L1, PD-L2,CTLA-4, B7-H3, B7-H4, CD40, OX40, CD137, among others.

In some aspects, the present chimeric agents are used in methods ofactivating a T cell, e.g., via the extracellular domain having an immunestimulatory signal.

In some aspects, the present chimeric agents are used in methods ofpreventing the cellular transmission of an immunosuppressive signal.

Combination Therapies and Conjugation

In embodiments, the invention provides for chimeric proteins and methodsthat further comprise administering an additional agent to a subject. Inembodiments, the invention pertains to co-administration and/orco-formulation. Any of the compositions described herein may beco-formulated and/or co-administered.

In embodiments, any chimeric protein described herein actssynergistically when co-administered with another agent and isadministered at doses that are lower than the doses commonly employedwhen such agents are used as monotherapy. In embodiments, any agentreferenced herein may be used in combination with any of the chimericproteins described herein.

In embodiments, any of the chimeric proteins disclosed herein may beco-administered with another chimeric protein disclosed herein. Withoutwishing to be bound by theory, it is believed that a combined regimeninvolving the administration of one or more chimeric proteins whichinduce an innate immune response and one or more chimeric proteins whichinduce an adaptive immune response may provide synergistic effects(e.g., synergistic anti-tumor effects).

In some aspects, there is provided a method of treating cancer,comprising administering to a subject in need thereof: (i) a firstchimeric protein comprising a general structure of Nterminus-(a)-(b)-(c)-C terminus, where: (a) is a first domain comprisingan extracellular domain of a Type I transmembrane protein, (b) is alinker comprising at least one cysteine residue capable of forming adisulfide bond, and (c) is a second domain comprising an extracellulardomain of a Type II transmembrane protein, and the first chimericprotein modulates the innate immune system; and (ii) a second chimericprotein comprising a general structure of N terminus-(a)-(b)-(c)-Cterminus, where (a) is a first domain comprising an extracellular domainof a Type I transmembrane protein, (b) is a linker comprising at leastone cysteine residue capable of forming a disulfide bond, and (c) is asecond domain comprising an extracellular domain of Type IItransmembrane protein, and the second chimeric protein modulates theadaptive immune system.

In some aspects, there is provided a method of treating cancer,comprising administering to a subject in need thereof: a second chimericprotein comprising a general structure of N terminus-(a)-(b)-(c)-Cterminus, where (a) is a first domain comprising an extracellular domainof a Type I transmembrane protein, (b) is a linker comprising at leastone cysteine residue capable of forming a disulfide bond, and (c) is asecond domain comprising an extracellular domain of Type IItransmembrane protein, and the second chimeric protein modulates theadaptive immune system, where the subject is undergoing or has undergonetreatment with a first chimeric protein comprising a general structureof N terminus-(a)-(b)-(c)-C terminus, where (a) is a first domaincomprising an extracellular domain of a Type I transmembrane protein,(b) is a linker comprising at least one cysteine residue capable offorming a disulfide bond, and (c) is a second domain comprising anextracellular domain of Type II transmembrane protein, and the firstchimeric protein modulates the innate immune system.

In embodiments, first chimeric protein is administered before the secondchimeric protein.

In embodiments, the first chimeric protein is administered after thesecond chimeric protein.

In embodiments, the first chimeric protein comprises at least one of:TIGIT, CSF1R, CD172a (SIRP1α), VSIG8, TIM3, 41BBL, CD40L, SIGLEC7,SIGLEC9 LIGHT.

In embodiments, the second chimeric protein comprises at least one of:PD-1, TIM3, VSIG8, CD172a (SIRP1α), OX40L, GITRL, TL1A, IL-2

In embodiments, the first chimeric protein and the second chimericprotein are independently selected from TIM3-Fc-OX40L, CD172a(SIRP1α)-Fc-CD40L, and CSF1R-Fc-CD40L.

In embodiments, TIM3-Fc-OX40L is administered before CD172a(SIRP1α)-Fc-CD40L. In embodiments, TIM3-Fc-OX40L is administered beforeCSF1R-Fc-CD40L. In embodiments, CD172a (SIRP1α)-Fc-CD40L is administeredbefore TIM3-Fc-OX40L. In embodiments, CSF1R-Fc-CD40L is administeredbefore TIM3-Fc-OX40L.

In embodiments, the first chimeric protein and/or the second chimericprotein causes activation of antigen presenting cells.

In embodiments, the first chimeric protein and/or the second chimericprotein enhances the ability of antigen presenting cells to presentantigen.

In embodiments, the first chimeric protein and/or the second chimericprotein provides a sustained immunomodulatory effect.

In embodiments, the first chimeric protein and/or the second chimericprotein prevents a tumor cell from transmitting an immunosuppressivesignal.

In embodiments, the second chimeric protein enhances tumor killingactivity by T cells.

In embodiments, any chimeric protein which induces an innate immuneresponse may be utilized in the present invention. In embodiments, anychimeric protein which induces an adaptive immune response may beutilized in the present invention. In an illustrative embodiment, achimeric protein which induce an innate immune response is a chimericprotein comprising the extracellular domain of CSF1R at the N-terminusand the extracellular domain of CD40L at the C-terminus. In anotherembodiment, a chimeric protein which induces an innate immune responseis a chimeric protein comprising the extracellular domain of SIRPα atthe N-terminus and the extracellular domain of CD40L at the C-terminus.In an illustrative embodiment, a chimeric protein which induce anadaptive immune response is a chimeric protein comprising theextracellular domain of PD-1 at the N-terminus and the extracellulardomain of OX40L at the C-terminus. In another embodiment, a chimericprotein which induces an adaptive immune response is a chimeric proteincomprising the extracellular domain of VSIG8 at the N-terminus and theextracellular domain of OX40L at the C-terminus.

In embodiments, the present invention relates to the co-administrationof a first chimeric protein, e.g., which induces an innate immuneresponse, and a second chimeric protein, e.g., which induces an adaptiveimmune response. In such embodiments, the first chimeric protein may beadministered before, concurrently with, or subsequent to administrationof the second chimeric protein. For example, the chimeric proteins maybe administered 1 minute apart, 10 minutes apart, 30 minutes apart, lessthan 1 hour apart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hoursto 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 11 hoursapart, 11 hours to 12 hours apart, 1 day apart, 2 days apart, 3 daysapart, 4 days apart, 5 days apart, 6 days apart, 1 week apart, 2 weeksapart, 3 weeks apart, or 4 weeks apart. In an illustrative embodiment,the first chimeric protein and the second chimeric protein areadministered 1 week apart, or administered on alternate weeks (i.e.,administration of the first chimeric i.e., protein is followed 1 weeklater with administration of the second chimeric protein and so forth).

Any chimeric protein disclosed herein can be a first chimeric protein,as described herein; any chimeric protein disclosed herein can be asecond chimeric protein, as described herein.

In embodiments, a chimeric protein comprising an extracellular domain ofTIM3 and an extracellular domain of OX40L is co-administered with achimeric protein comprising an extracellular domain of CD172a (SIRPα)and an extracellular domain of CD40L. In embodiments the chimericprotein comprising an extracellular domain of TIM3 and an extracellulardomain of OX40L is administered before the chimeric protein comprisingan extracellular domain of CD172a (SIRPα) and an extracellular domain ofCD40L. In embodiments the chimeric protein comprising an extracellulardomain of TIM3 and an extracellular domain of OX40L is administeredafter the chimeric protein comprising an extracellular domain of CD172a(SIRPα) and an extracellular domain of CD40L.

In embodiments, a chimeric protein comprising an extracellular domain ofTIM3 and an extracellular domain of OX40L is co-administered with achimeric protein comprising an extracellular domain of CSF1R and anextracellular domain of CD40L. In embodiments the chimeric proteincomprising an extracellular domain of TIM3 and an extracellular domainof OX40L is administered before the chimeric protein comprising anextracellular domain of CSF1R and an extracellular domain of CD40L. Inembodiments the chimeric protein comprising an extracellular domain ofTIM3 and an extracellular domain of OX40L is administered after thechimeric protein comprising an extracellular domain of CSF1R and anextracellular domain of CD40L. In embodiments, co-administrationincludes twice administering the same chimeric protein with the firstadministering and the second administering separated in time. Forexample, the first administering and the second administering may be 1minute apart, 10 minutes apart, 30 minutes apart, less than 1 hourapart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart,3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hoursapart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 11hours to 12 hours apart, 1 day apart, 2 days apart, 3 days apart, 4 daysapart, 5 days apart, 6 days apart, 1 week apart, 2 weeks apart, 3 weeksapart, or 4 weeks apart.

In embodiments, inclusive of, without limitation, cancer applications,the present invention pertains to chemotherapeutic agents as additionalagents. Examples of chemotherapeutic agents include, but are not limitedto, alkylating agents such as thiotepa and CYTOXAN cyclosphosphamide;alkyl sulfonates such as busulfan, improsulfan and piposulfan;aziridines such as benzodopa, carboquone, meturedopa, and uredopa;ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(e.g., bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; cally statin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (e.g., cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogues, KW-2189 andCB 1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gammall and calicheamicinomegall (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCINdoxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as minoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; demecolcine; diaziquone;elformithine; elliptinium acetate; an epothilone; etoglucid; galliumnitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such asmaytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOLpaclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANECremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, 111), andTAXOTERE doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; NAVELBINE. vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar,CPT-11) (including the treatment regimen of irinotecan with 5-FU andleucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;leucovorin (LV); oxaliplatin, including the oxaliplatin treatmentregimen (FOLFOX); lapatinib (TYKERB); inhibitors of PKC-α, Raf, H-Ras,EGFR (e.g., erlotinib (Tarceva)) and VEGF-A that reduce cellproliferation and pharmaceutically acceptable salts, acids orderivatives of any of the above. In addition, the methods of treatmentcan further include the use of radiation. In addition, the methods oftreatment can further include the use of photodynamic therapy.

In embodiments, inclusive of, without limitation, cancer applications,the present additional agent is one or more immune-modulating agentsselected from an agent that blocks, reduces and/or inhibits PD-1 andPD-L1 or PD-L2 and/or the binding of PD-1 with PD-L1 or PD-L2 (by way ofnon-limiting example, one or more of nivolumab (ONO-4538/BMS-936558,MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, Merck),MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), atezolizumab(TECENTRIQ, GENENTECH), MPDL328OA (ROCHE)), an agent that increasesand/or stimulates CD137 (4-1BB) and/or the binding of CD137 (4-1BB) withone or more of 4-1BB ligand (by way of non-limiting example, urelumab(BMS-663513 and anti-4-1BB antibody), and an agent that blocks, reducesand/or inhibits the activity of CTLA-4 and/or the binding of CTLA-4 withone or more of AP2M1, CD80, CD86, SHP-2, and PPP2R5A and/or the bindingof OX40 with OX40L (by way of non-limiting example GBR 830 (GLENMARK),MED16469 (MEDIMMUNE).

In embodiments, inclusive of, without limitation, infectious diseaseapplications, the present invention pertains to anti-infectives asadditional agents. In embodiments, the anti-infective is an anti-viralagent including, but not limited to, Abacavir, Acyclovir, Adefovir,Amprenavir, Atazanavir, Cidofovir, Darunavir, Delavirdine, Didanosine,Docosanol, Efavirenz, Elvitegravir, Emtricitabine, Enfuvirtide,Etravirine, Famciclovir, and Foscarnet. In embodiments, theanti-infective is an anti-bacterial agent including, but not limited to,cephalosporin antibiotics (cephalexin, cefuroxime, cefadroxil,cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin, cefprozil, andceftobiprole); fluoroquinolone antibiotics (cipro, Levaquin, floxin,tequin, avelox, and norflox); tetracycline antibiotics (tetracycline,minocycline, oxytetracycline, and doxycycline); penicillin antibiotics(amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin,vancomycin, and methicillin); monobactam antibiotics (aztreonam); andcarbapenem antibiotics (ertapenem, doripenem, imipenem/cilastatin, andmeropenem). In embodiments, the anti-infectives include anti-malarialagents (e.g., chloroquine, quinine, mefloquine, primaquine, doxycycline,artemether/lumefantrine, atovaquone/proguanil andsulfadoxine/pyrimethamine), metronidazole, tinidazole, ivermectin,pyrantel pamoate, and albendazole.

In embodiments, inclusive, without limitation, of autoimmuneapplications, the additional agent is an immunosuppressive agent. Inembodiments, the immunosuppressive agent is an anti-inflammatory agentsuch as a steroidal anti-inflammatory agent or a non-steroidalanti-inflammatory agent (NSAID). Steroids, particularly the adrenalcorticosteroids and their synthetic analogues, are well known in theart. Examples of corticosteroids useful in the present inventioninclude, without limitation, hydroxyltriamcinolone, alpha-methyldexamethasone, beta-methyl betamethasone, beclomethasone dipropionate,betamethasone benzoate, betamethasone dipropionate, betamethasonevalerate, clobetasol valerate, desonide, desoxymethasone, dexamethasone,diflorasone diacetate, diflucortolone valerate, fluadrenolone,fluclorolone acetonide, flumethasone pivalate, fluosinolone acetonide,fluocinonide, flucortine butylester, fluocortolone, fluprednidene(fluprednylidene) acetate, flurandrenolone, halcinonide, hydrocortisoneacetate, hydrocortisone butyrate, methylprednisolone, triamcinoloneacetonide, cortisone, cortodoxone, flucetonide, fludrocortisone,difluorosone diacetate, fluradrenolone acetonide, medrysone, amcinafel,amcinafide, betamethasone and the balance of its esters,chloroprednisone, clocortelone, clescinolone, dichlorisone,difluprednate, flucloronide, flunisolide, fluoromethalone, fluperolone,fluprednisolone, hydrocortisone, meprednisone, paramethasone,prednisolone, prednisone, beclomethasone dipropionate. (NSAIDS) that maybe used in the present invention, include but are not limited to,salicylic acid, acetyl salicylic acid, methyl salicylate, glycolsalicylate, salicylmides, benzyl-2,5-diacetoxybenzoic acid, ibuprofen,fulindac, naproxen, ketoprofen, etofenamate, phenylbutazone, andindomethacin. In embodiments, the immunosupressive agent may becytostatics such as alkylating agents, antimetabolites (e.g.,azathioprine, methotrexate), cytotoxic antibiotics, antibodies (e.g.,basiliximab, daclizumab, and muromonab), anti-immunophilins (e.g.,cyclosporine, tacrolimus, sirolimus), inteferons, opioids, TNF bindingproteins, mycophenolates, and small biological agents (e.g., fingolimod,myriocin).

In embodiments, the chimeric proteins (and/or additional agents)described herein, include derivatives that are modified, i.e., by thecovalent attachment of any type of molecule to the composition such thatcovalent attachment does not prevent the activity of the composition.For example, but not by way of limitation, derivatives includecomposition that have been modified by, inter alia, glycosylation,lipidation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Any ofnumerous chemical modifications can be carried out by known techniques,including, but not limited to specific chemical cleavage, acetylation,formylation, metabolic synthesis of turicamycin, etc. Additionally, thederivative can contain one or more non-classical amino acids. In stillother embodiments, the chimeric proteins (and/or additional agents)described herein further comprise a cytotoxic agent, comprising, inillustrative embodiments, a toxin, a chemotherapeutic agent, aradioisotope, and an agent that causes apoptosis or cell death. Suchagents may be conjugated to a composition described herein.

The chimeric proteins (and/or additional agents) described herein maythus be modified post-translationally to add effector moieties such aschemical linkers, detectable moieties such as for example fluorescentdyes, enzymes, substrates, bioluminescent materials, radioactivematerials, and chemiluminescent moieties, or functional moieties such asfor example streptavidin, avidin, biotin, a cytotoxin, a cytotoxicagent, and radioactive materials.

Formulations

The chimeric proteins (and/or additional agents) described herein canpossess a sufficiently basic functional group, which can react with aninorganic or organic acid, or a carboxyl group, which can react with aninorganic or organic base, to form a pharmaceutically acceptable salt. Apharmaceutically acceptable acid addition salt is formed from apharmaceutically acceptable acid, as is well known in the art. Suchsalts include the pharmaceutically acceptable salts listed in, forexample, Journal of Pharmaceutical Science, 66, 2-19 (1977) and TheHandbook of Pharmaceutical Salts; Properties, Selection, and Use. P. H.Stahl and C. G. Wermuth (eds.), Verlag, Zurich (Switzerland) 2002, whichare hereby incorporated by reference in their entirety.

In embodiments, the compositions described herein are in the form of apharmaceutically acceptable salt.

Further, any chimeric protein (and/or additional agents) describedherein can be administered to a subject as a component of a compositionthat comprises a pharmaceutically acceptable carrier or vehicle. Suchcompositions can optionally comprise a suitable amount of apharmaceutically acceptable excipient so as to provide the form forproper administration. Pharmaceutical excipients can be liquids, such aswater and oils, including those of petroleum, animal, vegetable, orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. The pharmaceutical excipients can be, for example,saline, gum acacia, gelatin, starch paste, talc, keratin, colloidalsilica, urea and the like. In addition, auxiliary, stabilizing,thickening, lubricating, and coloring agents can be used. In oneembodiment, the pharmaceutically acceptable excipients are sterile whenadministered to a subject. Water is a useful excipient when any agentdescribed herein is administered intravenously. Saline solutions andaqueous dextrose and glycerol solutions can also be employed as liquidexcipients, specifically for injectable solutions. Suitablepharmaceutical excipients also include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. Any agent describedherein, if desired, can also comprise minor amounts of wetting oremulsifying agents, or pH buffering agents.

In embodiments, the compositions described herein are resuspended in asaline buffer (including, without limitation TBS, PBS, and the like).

In embodiments, the chimeric proteins may by conjugated and/or fusedwith another agent to extend half-life or otherwise improvepharmacodynamic and pharmacokinetic properties. In embodiments, thechimeric proteins may be fused or conjugated with one or more of PEG,XTEN (e.g., as rPEG), polysialic acid (POLYXEN), albumin (e.g., humanserum albumin or HAS), elastin-like protein (ELP), PAS, HAP, GLK, CTP,transferrin, and the like. In embodiments, each of the individualchimeric proteins is fused to one or more of the agents described inBioDrugs (2015) 29:215-239, the entire contents of which are herebyincorporated by reference.

Administration, Dosing, and Treatment Regimens

The present invention includes the described chimeric protein (and/oradditional agents) in various formulations. Any chimeric protein (and/oradditional agents) described herein can take the form of solutions,suspensions, emulsion, drops, tablets, pills, pellets, capsules,capsules containing liquids, powders, sustained-release formulations,suppositories, emulsions, aerosols, sprays, suspensions, or any otherform suitable for use. DNA or RNA constructs encoding the proteinsequences may also be used. In one embodiment, the composition is in theform of a capsule (see, e.g., U.S. Pat. No. 5,698,155). Other examplesof suitable pharmaceutical excipients are described in Remington'sPharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed.1995), incorporated herein by reference.

Where necessary, the formulations comprising the chimeric protein(and/or additional agents) can also include a solubilizing agent. Also,the agents can be delivered with a suitable vehicle or delivery deviceas known in the art. Combination therapies outlined herein can beco-delivered in a single delivery vehicle or delivery device.Compositions for administration can optionally include a localanesthetic such as, for example, lignocaine to lessen pain at the siteof the injection.

The formulations comprising the chimeric protein (and/or additionalagents) of the present invention may conveniently be presented in unitdosage forms and may be prepared by any of the methods well known in theart of pharmacy. Such methods generally include the step of bringingtherapeutic agents into association with a carrier, which constitutesone or more accessory ingredients. Typically, the formulations areprepared by uniformly and intimately bringing therapeutic agent intoassociation with a liquid carrier, a finely divided solid carrier, orboth, and then, if necessary, shaping the product into dosage forms ofthe desired formulation (e.g., wet or dry granulation, powder blends,etc., followed by tableting using conventional methods known in the art)

In one embodiment, any chimeric protein (and/or additional agents)described herein is formulated in accordance with routine procedures asa composition adapted for a mode of administration described herein.

Routes of administration include, for example: intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, oral, sublingual, intranasal, intracerebral, intravaginal,transdermal, rectally, by inhalation, or topically, particularly to theears, nose, eyes, or skin. In embodiments, the administering is effectedorally or by parenteral injection. In most instances, administrationresults in the release of any agent described herein into thebloodstream.

Any chimeric protein (and/or additional agents) described herein can beadministered orally. Such chimeric proteins (and/or additional agents)can also be administered by any other convenient route, for example, byintravenous infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and can be administered together with anotherbiologically active agent. Administration can be systemic or local.Various delivery systems are known, e.g., encapsulation in liposomes,microparticles, microcapsules, capsules, etc., and can be used toadminister.

In specific embodiments, it may be desirable to administer locally tothe area in need of treatment. In one embodiment, for instance in thetreatment of cancer, the chimeric protein (and/or additional agents) areadministered in the tumor microenvironment (e.g., cells, molecules,extracellular matrix and/or blood vessels that surround and/or feed atumor cell, inclusive of, for example, tumor vasculature;tumor-infiltrating lymphocytes; fibroblast reticular cells; endothelialprogenitor cells (EPC); cancer-associated fibroblasts; pericytes; otherstromal cells; components of the extracellular matrix (ECM); dendriticcells; antigen presenting cells; T-cells; regulatory T cells;macrophages; neutrophils; and other immune cells located proximal to atumor) or lymph node and/or targeted to the tumor microenvironment orlymph node. In embodiments, for instance in the treatment of cancer, thechimeric protein (and/or additional agents) are administeredintratumorally.

In the various embodiments, the present chimeric protein allows for adual effect that provides less side effects than are seen inconventional immunotherapy (e.g., treatments with one or more of OPDIVO,KEYTRUDA, YERVOY, and TECENTRIQ). For example, the present chimericproteins reduce or prevent commonly observed immune-related adverseevents that affect various tissues and organs including the skin, thegastrointestinal tract, the kidneys, peripheral and central nervoussystem, liver, lymph nodes, eyes, pancreas, and the endocrine system;such as hypophysitis, colitis, hepatitis, pneumonitis, rash, andrheumatic disease. Further, the present local administration, e.g.,intratumorally, obviate adverse event seen with standard systemicadministration, e.g., IV infusions, as are used with conventionalimmunotherapy (e.g., treatments with one or more of OPDIVO, KEYTRUDA,YERVOY, and TECENTRIQ).

Dosage forms suitable for parenteral administration (e.g., intravenous,intramuscular, intraperitoneal, subcutaneous and intra-articularinjection and infusion) include, for example, solutions, suspensions,dispersions, emulsions, and the like. They may also be manufactured inthe form of sterile solid compositions (e.g., lyophilized composition),which can be dissolved or suspended in sterile injectable mediumimmediately before use. They may contain, for example, suspending ordispersing agents known in the art.

The dosage of any chimeric protein (and/or additional agents) describedherein as well as the dosing schedule can depend on various parameters,including, but not limited to, the disease being treated, the subject'sgeneral health, and the administering physician's discretion. Anychimeric protein described herein, can be administered prior to (e.g., 5minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before),concurrently with, or subsequent to (e.g., 5 minutes, 15 minutes, 30minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks,5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of anadditional agent, to a subject in need thereof. In embodiments anychimeric protein and additional agent described herein are administered1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hourapart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart,3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hoursapart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 11hours to 12 hours apart, 1 day apart, 2 days apart, 3 days apart, 4 daysapart, 5 days apart, 6 days apart, 1 week apart, 2 weeks apart, 3 weeksapart, or 4 weeks apart.

In embodiments, the present invention relates to the co-administrationof a chimeric protein which induces an innate immune response andanother chimeric protein which induces an adaptive immune response. Insuch embodiments, the chimeric protein which induces an innate immuneresponse may be administered before, concurrently with, or subsequent toadministration of the chimeric protein which induces an adaptive immuneresponse. For example, the chimeric proteins may be administered 1minute apart, 10 minutes apart, 30 minutes apart, less than 1 hourapart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart,3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hoursapart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 11hours to 12 hours apart, 1 day apart, 2 days apart, 3 days apart, 4 daysapart, 5 days apart, 6 days apart, 1 week apart, 2 weeks apart, 3 weeksapart, or 4 weeks apart. In an illustrative embodiment, the chimericprotein which induces an innate immune response and the chimeric proteinwhich induces an adaptive response are administered 1 week apart, oradministered on alternate weeks (i.e., administration of the chimericprotein inducing an innate immune response is followed 1 week later withadministration of the chimeric protein which induces an adaptive immuneresponse and so forth).

The dosage of any chimeric protein (and/or additional agents) describedherein can depend on several factors including the severity of thecondition, whether the condition is to be treated or prevented, and theage, weight, and health of the subject to be treated. Additionally,pharmacogenomic (the effect of genotype on the pharmacokinetic,pharmacodynamic or efficacy profile of a therapeutic) information abouta particular subject may affect dosage used. Furthermore, the exactindividual dosages can be adjusted somewhat depending on a variety offactors, including the specific combination of the agents beingadministered, the time of administration, the route of administration,the nature of the formulation, the rate of excretion, the particulardisease being treated, the severity of the disorder, and the anatomicallocation of the disorder. Some variations in the dosage can be expected.

For administration of any chimeric protein (and/or additional agents)described herein by parenteral injection, the dosage may be about 0.1 mgto about 250 mg per day, about 1 mg to about 20 mg per day, or about 3mg to about 5 mg per day. Generally, when orally or parenterallyadministered, the dosage of any agent described herein may be about 0.1mg to about 1500 mg per day, or about 0.5 mg to about 10 mg per day, orabout 0.5 mg to about 5 mg per day, or about 200 to about 1,200 mg perday (e.g., about 200 mg, about 300 mg, about 400 mg, about 500 mg, about600 mg, about 700 mg, about 800 mg, about 900 mg, about 1,000 mg, about1,100 mg, about 1,200 mg per day).

In embodiments, administration of the chimeric protein (and/oradditional agents) described herein is by parenteral injection at adosage of about 0.1 mg to about 1500 mg per treatment, or about 0.5 mgto about 10 mg per treatment, or about 0.5 mg to about 5 mg pertreatment, or about 200 to about 1,200 mg per treatment (e.g., about 200mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700mg, about 800 mg, about 900 mg, about 1,000 mg, about 1,100 mg, about1,200 mg per treatment).

In embodiments, a suitable dosage of the chimeric protein (and/oradditional agents) is in a range of about 0.01 mg/kg to about 100 mg/kgof body weight, or about 0.01 mg/kg to about 10 mg/kg of body weight ofthe subject, for example, about 0.01 mg/kg, about 0.02 mg/kg, about 0.03mg/kg, about 0.04 mg/kg, about 0.05 mg/kg, about 0.06 mg/kg, about 0.07mg/kg, about 0.08 mg/kg, about 0.09 mg/kg, about 0.1 mg/kg, about 0.2mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1 mg/kg,about 1.1 mg/kg, about 1.2 mg/kg, about 1.3 mg/kg, about 1.4 mg/kg,about 1.5 mg/kg, about 1.6 mg/kg, about 1.7 mg/kg, about 1.8 mg/kg, 1.9mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kgbody weight, inclusive of all values and ranges therebetween.

In another embodiment, delivery can be in a vesicle, in particular aliposome (see Langer, 1990, Science 249:1527-1533; Treat et al., inLiposomes in therapy of Infectious Disease and Cancer, Lopez-Beresteinand Fidler (eds.), Liss, New York, pp. 353-365 (1989).

Any chimeric protein (and/or additional agents) described herein can beadministered by controlled-release or sustained-release means or bydelivery devices that are well known to those of ordinary skill in theart. Examples include, but are not limited to, those described in U.S.Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719;5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476;5,354,556; and 5,733,556, each of which is incorporated herein byreference in its entirety. Such dosage forms can be useful for providingcontrolled- or sustained-release of one or more active ingredientsusing, for example, hydropropylmethyl cellulose, other polymer matrices,gels, permeable membranes, osmotic systems, multilayer coatings,microparticles, liposomes, microspheres, or a combination thereof toprovide the desired release profile in varying proportions. Controlled-or sustained-release of an active ingredient can be stimulated byvarious conditions, including but not limited to, changes in pH, changesin temperature, stimulation by an appropriate wavelength of light,concentration or availability of enzymes, concentration or availabilityof water, or other physiological conditions or compounds.

In another embodiment, polymeric materials can be used (see MedicalApplications of Controlled Release, Langer and Wise (eds.), CRC Pres.,Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug ProductDesign and Performance, Smolen and Ball (eds.), Wiley, New York (1984);Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61;see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann.Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105).

In another embodiment, a controlled-release system can be placed inproximity of the target area to be treated, thus requiring only afraction of the systemic dose (see, e.g., Goodson, in MedicalApplications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).Other controlled-release systems discussed in the review by Langer,1990, Science 249:1527-1533) may be used.

Administration of any chimeric protein (and/or additional agents)described herein can, independently, be one to four times daily or oneto four times per month or one to six times per year or once every two,three, four or five years. Administration can be for the duration of oneday or one month, two months, three months, six months, one year, twoyears, three years, and may even be for the life of the subject.

The dosage regimen utilizing any chimeric protein (and/or additionalagents) described herein can be selected in accordance with a variety offactors including type, species, age, weight, sex and medical conditionof the subject; the severity of the condition to be treated; the routeof administration; the renal or hepatic function of the subject; thepharmacogenomic makeup of the individual; and the specific compound ofthe invention employed. Any chimeric protein (and/or additional agents)described herein can be administered in a single daily dose, or thetotal daily dosage can be administered in divided doses of two, three orfour times daily. Furthermore, any chimeric protein (and/or additionalagents) described herein can be administered continuously rather thanintermittently throughout the dosage regimen.

Cells and Nucleic Acids

In embodiments, the present invention provides an expression vector,comprising a nucleic acid encoding the chimeric protein describedherein. In embodiments, the expression vector comprises DNA or RNA. Inembodiments, the expression vector is a mammalian expression vector.

Both prokaryotic and eukaryotic vectors can be used for expression ofthe chimeric protein. Prokaryotic vectors include constructs based on E.coli sequences (see, e.g., Makrides, Microbiol Rev 1996, 60:512-538).Non-limiting examples of regulatory regions that can be used forexpression in E. coli include lac, trp, Ipp, phoA, recA, tac, T3, T7 andλP_(L). Non-limiting examples of prokaryotic expression vectors mayinclude the λgt vector series such as λgt11 (Huynh et al., in “DNACloning Techniques, Vol. I: A Practical Approach,” 1984, (D. Glover,ed.), pp. 49-78, IRL Press, Oxford), and the pET vector series (Studieret al., Methods Enzymol 1990, 185:60-89). Prokaryotic host-vectorsystems cannot perform much of the post-translational processing ofmammalian cells, however. Thus, eukaryotic host-vector systems may beparticularly useful. A variety of regulatory regions can be used forexpression of the chimeric proteins in mammalian host cells. Forexample, the SV40 early and late promoters, the cytomegalovirus (CMV)immediate early promoter, and the Rous sarcoma virus long terminalrepeat (RSV-LTR) promoter can be used. Inducible promoters that may beuseful in mammalian cells include, without limitation, promotersassociated with the metallothionein II gene, mouse mammary tumor virusglucocorticoid responsive long terminal repeats (MMTV-LTR), theβ-interferon gene, and the hsp70 gene (see, Williams et al., Cancer Res1989, 49:2735-42; and Taylor et al., Mol Cell Biol 1990, 10:165-75).Heat shock promoters or stress promoters also may be advantageous fordriving expression of the chimeric proteins in recombinant host cells.

In embodiments, expression vectors of the invention comprise a nucleicacid encoding the chimeric proteins (and/or additional agents), or acomplement thereof, operably linked to an expression control region, orcomplement thereof, that is functional in a mammalian cell. Theexpression control region is capable of driving expression of theoperably linked blocking and/or stimulating agent encoding nucleic acidsuch that the blocking and/or stimulating agent is produced in a humancell transformed with the expression vector.

Expression control regions are regulatory polynucleotides (sometimesreferred to herein as elements), such as promoters and enhancers, thatinfluence expression of an operably linked nucleic acid. An expressioncontrol region of an expression vector of the invention is capable ofexpressing operably linked encoding nucleic acid in a human cell. In anembodiment, the cell is a tumor cell. In another embodiment, the cell isa non-tumor cell. In an embodiment, the expression control regionconfers regulatable expression to an operably linked nucleic acid. Asignal (sometimes referred to as a stimulus) can increase or decreaseexpression of a nucleic acid operably linked to such an expressioncontrol region. Such expression control regions that increase expressionin response to a signal are often referred to as inducible. Suchexpression control regions that decrease expression in response to asignal are often referred to as repressible. Typically, the amount ofincrease or decrease conferred by such elements is proportional to theamount of signal present; the greater the amount of signal, the greaterthe increase or decrease in expression.

In an embodiment, the present invention contemplates the use ofinducible promoters capable of effecting high level of expressiontransiently in response to a cue. For example, when in the proximity ofa tumor cell, a cell transformed with an expression vector for thechimeric protein (and/or additional agents) comprising such anexpression control sequence is induced to transiently produce a highlevel of the agent by exposing the transformed cell to an appropriatecue. Illustrative inducible expression control regions include thosecomprising an inducible promoter that is stimulated with a cue such as asmall molecule chemical compound. Particular examples can be found, forexample, in U.S. Pat. Nos. 5,989,910, 5,935,934, 6,015,709, and6,004,941, each of which is incorporated herein by reference in itsentirety.

Expression control regions and locus control regions include full-lengthpromoter sequences, such as native promoter and enhancer elements, aswell as subsequences or polynucleotide variants which retain all or partof full-length or non-variant function. As used herein, the term“functional” and grammatical variants thereof, when used in reference toa nucleic acid sequence, subsequence or fragment, means that thesequence has one or more functions of native nucleic acid sequence(e.g., non-variant or unmodified sequence).

As used herein, “operable linkage” refers to a physical juxtaposition ofthe components so described as to permit them to function in theirintended manner. In the example of an expression control element inoperable linkage with a nucleic acid, the relationship is such that thecontrol element modulates expression of the nucleic acid. Typically, anexpression control region that modulates transcription is juxtaposednear the 5′ end of the transcribed nucleic acid (i.e., “upstream”).Expression control regions can also be located at the 3′ end of thetranscribed sequence (i.e., “downstream”) or within the transcript(e.g., in an intron). Expression control elements can be located at adistance away from the transcribed sequence (e.g., 100 to 500, 500 to1000, 2000 to 5000, or more nucleotides from the nucleic acid). Aspecific example of an expression control element is a promoter, whichis usually located 5′ of the transcribed sequence. Another example of anexpression control element is an enhancer, which can be located 5′ or 3′of the transcribed sequence, or within the transcribed sequence.

Expression systems functional in human cells are well known in the art,and include viral systems. Generally, a promoter functional in a humancell is any DNA sequence capable of binding mammalian RNA polymerase andinitiating the downstream (3′) transcription of a coding sequence intomRNA. A promoter will have a transcription initiating region, which isusually placed proximal to the 5′ end of the coding sequence, andtypically a TATA box located 25-30 base pairs upstream of thetranscription initiation site. The TATA box is thought to direct RNApolymerase II to begin RNA synthesis at the correct site. A promoterwill also typically contain an upstream promoter element (enhancerelement), typically located within 100 to 200 base pairs upstream of theTATA box. An upstream promoter element determines the rate at whichtranscription is initiated and can act in either orientation. Ofparticular use as promoters are the promoters from mammalian viralgenes, since the viral genes are often highly expressed and have a broadhost range. Examples include the SV40 early promoter, mouse mammarytumor virus LTR promoter, adenovirus major late promoter, herpes simplexvirus promoter, and the CMV promoter.

Typically, transcription termination and polyadenylation sequencesrecognized by mammalian cells are regulatory regions located 3′ to thetranslation stop codon and thus, together with the promoter elements,flank the coding sequence. The 3′ terminus of the mature mRNA is formedby site-specific post-translational cleavage and polyadenylation.Examples of transcription terminator and polyadenylation signals includethose derived from SV40. Introns may also be included in expressionconstructs.

There are a variety of techniques available for introducing nucleicacids into viable cells. Techniques suitable for the transfer of nucleicacid into mammalian cells in vitro include the use of liposomes,electroporation, microinjection, cell fusion, polymer-based systems,DEAE-dextran, viral transduction, the calcium phosphate precipitationmethod, etc. For in vivo gene transfer, a number of techniques andreagents may also be used, including liposomes; natural polymer-baseddelivery vehicles, such as chitosan and gelatin; viral vectors are alsosuitable for in vivo transduction. In some situations, it is desirableto provide a targeting agent, such as an antibody or ligand specific fora tumor cell surface membrane protein. Where liposomes are employed,proteins which bind to a cell surface membrane protein associated withendocytosis may be used for targeting and/or to facilitate uptake, e.g.,capsid proteins or fragments thereof tropic for a particular cell type,antibodies for proteins which undergo internalization in cycling,proteins that target intracellular localization and enhanceintracellular half-life. The technique of receptor-mediated endocytosisis described, for example, by Wu et al., J. Biol. Chem. 262, 4429-4432(1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414(1990).

Where appropriate, gene delivery agents such as, e.g., integrationsequences can also be employed. Numerous integration sequences are knownin the art (see, e.g., Nunes-Duby et al., Nucleic Acids Res. 26:391-406,1998; Sadwoski, J. Bacteriol., 165:341-357, 1986; Bestor, Cell,122(3):322-325, 2005; Plasterk et al., TIG 15:326-332, 1999; Kootstra etal., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). These includerecombinases and transposases. Examples include Cre (Sternberg andHamilton, J. Mol. Biol., 150:467-486, 1981), lambda (Nash, Nature, 247,543-545, 1974), Flp (Broach, et al., Cell, 29:227-234, 1982), R(Matsuzaki, et al., J. Bacteriology, 172:610-618, 1990), cpC31 (see,e.g., Groth et al., J. Mol. Biol. 335:667-678, 2004), sleeping beauty,transposases of the mariner family (Plasterk et al., supra), andcomponents for integrating viruses such as MV, retroviruses, andantiviruses having components that provide for virus integration such asthe LTR sequences of retroviruses or lentivirus and the ITR sequences ofAAV (Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). Inaddition, direct and targeted genetic integration strategies may be usedto insert nucleic acid sequences encoding the chimeric fusion proteinsincluding CRISPR/CAS9, zinc finger, TALEN, and meganuclease gene-editingtechnologies.

In one aspect, the invention provides expression vectors for theexpression of the chimeric proteins (and/or additional agents) that areviral vectors. Many viral vectors useful for gene therapy are known(see, e.g., Lundstrom, Trends Biotechnol., 21: 1 17, 122, 2003.Illustrative viral vectors include those selected from Antiviruses (LV),retroviruses (RV), adenoviruses (AV), adeno-associated viruses (MV), anda viruses, though other viral vectors may also be used. For in vivouses, viral vectors that do not integrate into the host genome aresuitable for use, such as a viruses and adenoviruses. Illustrative typesof a viruses include Sindbis virus, Venezuelan equine encephalitis (VEE)virus, and Semliki Forest virus (SFV). For in vitro uses, viral vectorsthat integrate into the host genome are suitable, such as retroviruses,AAV, and Antiviruses. In one embodiment, the invention provides methodsof transducing a human cell in vivo, comprising contacting a solid tumorin vivo with a viral vector of the invention.

In embodiments, the present invention provides a host cell, comprisingthe expression vector comprising the chimeric protein described herein.

Expression vectors can be introduced into host cells for producing thepresent chimeric proteins. Cells may be cultured in vitro or geneticallyengineered, for example. Useful mammalian host cells include, withoutlimitation, cells derived from humans, monkeys, and rodents (see, forexample, Kriegler in “Gene Transfer and Expression: A LaboratoryManual,” 1990, New York, Freeman & Co.). These include monkey kidneycell lines transformed by SV40 (e.g., COS-7, ATCC CRL 1651); humanembryonic kidney lines (e.g., 293, 293-EBNA, or 293 cells subcloned forgrowth in suspension culture, Graham et al., J Gen Virol 1977, 36:59);baby hamster kidney cells (e.g., BHK, ATCC CCL 10); Chinese hamsterovary-cells-DHFR (e.g., CHO, Urlaub and Chasin, Proc Natl Acad Sci USA1980, 77:4216); DG44 CHO cells, CHO-K1 cells, mouse Sertoli cells(Mather, Biol Reprod 1980, 23:243-251); mouse fibroblast cells (e.g.,NIH-3T3), monkey kidney cells (e.g., CV1 ATCC CCL 70); African greenmonkey kidney cells. (e.g., VERO-76, ATCC CRL-1587); human cervicalcarcinoma cells (e.g., HELA, ATCC CCL 2); canine kidney cells (e.g.,MDCK, ATCC CCL 34); buffalo rat liver cells (e.g., BRL 3A, ATCC CRL1442); human lung cells (e.g., W138, ATCC CCL 75); human liver cells(e.g., Hep G2, HB 8065); and mouse mammary tumor cells (e.g., MMT060562, ATCC CCL51). Illustrative cancer cell types for expressing thechimeric proteins described herein include mouse fibroblast cell line,NIH3T3, mouse Lewis lung carcinoma cell line, LLC, mouse mastocytomacell line, P815, mouse lymphoma cell line, EL4 and its ovalbumintransfectant, E.G7, mouse melanoma cell line, B16F10, mouse fibrosarcomacell line, MC57, and human small cell lung carcinoma cell lines, SCLC #2and SCLC #7.

Host cells can be obtained from normal or affected subjects, includinghealthy humans, cancer patients, and patients with an infectiousdisease, private laboratory deposits, public culture collections such asthe American Type Culture Collection, or from commercial suppliers.

Cells that can be used for production of the present chimeric proteinsin vitro, ex vivo, and/or in vivo include, without limitation,epithelial cells, endothelial cells, keratinocytes, fibroblasts, musclecells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes,monocytes, macrophages, neutrophils, eosinophils, megakaryocytes,granulocytes; various stem or progenitor cells, in particularhematopoietic stem or progenitor cells (e.g., as obtained from bonemarrow), umbilical cord blood, peripheral blood, fetal liver, etc. Thechoice of cell type depends on the type of tumor or infectious diseasebeing treated or prevented, and can be determined by one of skill in theart.

Production and purification of Fc-containing macromolecules (such asmonoclonal antibodies) has become a standardized process, with minormodifications between products. For example, many Fc containingmacromolecules are produced by human embryonic kidney (HEK) cells (orvariants thereof) or Chinese Hamster Ovary (CHO) cells (or variantsthereof) or in some cases by bacterial or synthetic methods. Followingproduction, the Fc containing macromolecules that are secreted by HEK orCHO cells are purified through binding to Protein A columns andsubsequently ‘polished’ using various methods. Generally speaking,purified Fc containing macromolecules are stored in liquid form for someperiod of time, frozen for extended periods of time or in some caseslyophilized. In embodiments, production of the chimeric proteinscontemplated herein may have unique characteristics as compared totraditional Fc containing macromolecules. In certain examples, thechimeric proteins may be purified using specific chromatography resins,or using chromatography methods that do not depend upon Protein Acapture. In embodiments, the chimeric proteins may be purified in anoligomeric state, or in multiple oligomeric states, and enriched for aspecific oligomeric state using specific methods. Without being bound bytheory, these methods could include treatment with specific buffersincluding specified salt concentrations, pH and additive compositions.In other examples, such methods could include treatments that favor oneoligomeric state over another. The chimeric proteins obtained herein maybe additionally ‘polished’ using methods that are specified in the art.In embodiments, the chimeric proteins are highly stable and able totolerate a wide range of pH exposure (between pH 3-12), are able totolerate a large number of freeze/thaw stresses (greater than 3freeze/thaw cycles) and are able to tolerate extended incubation at hightemperatures (longer than 2 weeks at 40 degrees C.). In embodiments, thechimeric proteins are shown to remain intact, without evidence ofdegradation, deamidation, etc. under such stress conditions.

Subjects and/or Animals

In embodiments, the subject and/or animal is a mammal, e.g., a human,mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, ornon-human primate, such as a monkey, chimpanzee, or baboon. Inembodiments, the subject and/or animal is a non-mammal, such, forexample, a zebrafish. In embodiments, the subject and/or animal maycomprise fluorescently-tagged cells (with e.g., GFP). In embodiments,the subject and/or animal is a transgenic animal comprising afluorescent cell.

In embodiments, the subject and/or animal is a human. In embodiments,the human is a pediatric human. In embodiments, the human is an adulthuman. In embodiments, the human is a geriatric human. In embodiments,the human may be referred to as a patient.

In certain embodiments, the human has an age in a range of from about 0months to about 6 months old, from about 6 to about 12 months old, fromabout 6 to about 18 months old, from about 18 to about 36 months old,from about 1 to about 5 years old, from about 5 to about 10 years old,from about 10 to about 15 years old, from about 15 to about 20 yearsold, from about 20 to about 25 years old, from about 25 to about 30years old, from about 30 to about 35 years old, from about 35 to about40 years old, from about 40 to about 45 years old, from about 45 toabout 50 years old, from about 50 to about 55 years old, from about 55to about 60 years old, from about 60 to about 65 years old, from about65 to about 70 years old, from about 70 to about 75 years old, fromabout 75 to about 80 years old, from about 80 to about 85 years old,from about 85 to about 90 years old, from about 90 to about 95 years oldor from about 95 to about 100 years old.

In embodiments, the subject is a non-human animal, and therefore theinvention pertains to veterinary use. In a specific embodiment, thenon-human animal is a household pet. In another specific embodiment, thenon-human animal is a livestock animal.

Kits

The invention provides kits that can simplify the administration of anyagent described herein. An illustrative kit of the invention comprisesany composition described herein in unit dosage form. In one embodiment,the unit dosage form is a container, such as a pre-filled syringe, whichcan be sterile, containing any agent described herein and apharmaceutically acceptable carrier, diluent, excipient, or vehicle. Thekit can further comprise a label or printed instructions instructing theuse of any agent described herein. The kit may also include a lidspeculum, topical anesthetic, and a cleaning agent for theadministration location. The kit can also further comprise one or moreadditional agent described herein. In one embodiment, the kit comprisesa container containing an effective amount of a composition of theinvention and an effective amount of another composition, such thosedescribed herein.

Any aspect or embodiment described herein can be combined with any otheraspect or embodiment as disclosed herein

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1: In Silico Predicted Structure of Monomeric HumanCD172a (SIRPα)-Fc-CD40L Chimeric Protein

An in silico structure prediction of the monomeric human CD172a(SIRPα)-Fc-CD40L chimeric protein (SL-172154) having 792 amino acidresidues was generated, with a p-value 1.14×10⁻²¹. The molecular weightof the monomeric protein was predicted to be 88.1 kDa. A structure ofthe chimeric protein is provided in FIG. 2.

Specifically, the structure prediction revealed that 48 positions (6%)may be disordered. Secondary structure prediction of the entire sequenceof the chimeric protein showed that the protein has the composition of2% α-helix (H), 50% 3-sheet (E), and 47% coil (C). The GDT (globaldistance test) and uGDT (un-normalized GDT) for the absolute globalquality were also calculated for the chimeric protein to give an overalluGDT(GDT) of 429(54). The three-state prediction for solventaccessibility of the protein residues were 33% exposed (E), 48%intermediate (M), and 17% buried (B).

A schematic diagram illustrating a mechanism of action of the hCD172a(SIRPα)-Fc-CD40L chimeric protein for stimulating active tumordestruction is shown in FIG. 3. The chimeric protein may then “dangle”from the surface of the tumor cell, and the CD40L portion of thechimeric protein may then bind to CD40 expressed on the surface of the Tcell. This would result in replacement of an inhibitory hCD172a (SIRPα)signal with a co-stimulatory CD40L signal to enhance the anti-tumoractivity of T cells.

Many tumor types express high levels of membrane-bound CD47, which canbind to CD172a (SIRPα) on the surface of a macrophage, thereby inducinga ‘don't eat me’ signal that inhibits macrophage engulfment orphagocytosis of the tumor cell. Without wishing to be bound by theory,it is believed that the CD172a (SIRPα)-Fc-CD40L chimeric protein(SL-172154) binds tumor that express CD47, blocking its interaction withmacrophages, thereby allowing macrophage maturation andphagocytosis-mediated destruction of the tumor cells. This in turnresults in enhanced tumor-antigen release and cross-presentation back toother macrophages. Interestingly, the cross-presentation of antigensoccurs at the same time and in the same tumor microenvironmental contextas co-stimulation between macrophage/APC bound CD40 and CD40L from thechimeric protein. The CD172a (SIRPα)-Fc-CD40L chimeric protein thereforestimulates both active tumor destruction, and also immune recognition oftumor antigens, which are essential in programming a memory responsecapable of preventing relapse (FIG. 3).

Example 2: Characterization of Human CD172a (SIRPα)-Fc-CD40L ChimericProtein

A human CD172a (SIRPα)-Fc-CD40L_chimeric protein was constructed. Thechimeric protein was characterized by performing a Western blot analysisagainst each individual domain of the chimeric protein, i.e., viaanti-CD172a (SIRPα), anti-Fc, and anti-CD40L antibodies.

The Western blots indicated the presence of a dominant dimer band in thenon-reduced lanes (FIG. 4, lane 2 in each blot), which was reduced to aglycosylated monomeric band in the presence of the reducing agent,β-mercaptoethanol (FIG. 4, lane 3 in each blot). As shown in FIG. 4,lane 4 in each blot, the chimeric protein ran as a monomer at thepredicted molecular weight of 88.1 kDa in the presence of both areducing agent (β-mercaptoethanol) and an endoglycosidase (PNGase).

Example 3: Characterization of the Binding Affinity of the DifferentDomains of the CD172a(SIRPα)-Fc-CD40L Chimeric Protein Using ELISA

Functional ELISA (enzyme-linked immunosorbent assay) assays weredeveloped to demonstrate the binding affinity of the different domainsof the human CD172a (SIRPα)-Fc-CD40L chimeric protein to theirrespective binding partners. The CD172a (SIRPα) domain of the hCD172a(SIRPα)-Fc-CD40L chimeric protein was detected by capturing to aplate-bound recombinant human CD47 protein and detected via an HRPconjugated anti-human IgG antibody. Recombinant hCD172a (SIRPα)-Fcprotein was used to generate a standard curve. The Fc portion of thehCD172a (SIRPα)-Fc-CD40L chimeric protein was detected by capturing to aplate-bound human IgG and detected via an HRP conjugated anti-human IgGantibody (hIg). Recombinant hIg protein was used to generate a standardcurve. The CD40L domain of the hCD172a (SIRPα)-Fc-CD40L chimeric proteinwas detected by capturing to a plate-bound recombinant human CD40protein and detected via a CD40L-specific antibody. Recombinant hCD40Lprotein was used to generate a standard curve.

As shown in FIG. 6, the different domains of the hCD172a(SIRPα)-Fc-CD40L chimeric protein effectively interacted with theirrespective binding partners in a concentration-dependent manner and withhigh affinity. Moreover, as shown in the right panel of FIG. 6, thehCD172a (SIRPα)-Fc-CD40L chimeric protein is able to contemporaneouslybind to both CD40 and CD47. Nevertheless, it was observed that in ELISAassays, using the central Fc region to detect chimeric proteins tendedto underestimate the actual protein content in a sample. Therefore, lowlevel of the hCD172a (SIRPα)-Fc-CD40L_chimeric protein was detectedcompared to standard in this assay. Based on this data an EC₅₀ value forCD47 of 1.39 nM, for mCD172a (SIRPα) of 1.12 nM, and for contemporaneousCD47 and CD40 of 18.1 nm were calculated for the hCD172a(SIRPα)-Fc-CD40L chimeric protein.

Ex vivo cell binding assays were also utilized to assess the ability ofthe different domains of the CD172a (SIRPα)-Fc-CD40L chimeric protein tobind their respective binding partners. Here, a cell line was engineeredto overexpress human CD47 (i.e., HeLa/hCD47) and a cell line wasengineered to overexpress murine CD40 (i.e., CHOK1/mCD40).

Human CD172a (SIRPα)-Fc-CD40L chimeric protein or murine CD172a(SIRPα)-Fc-CD40L chimeric protein was incubated with the parental andover-expressing cell lines for 2 hours. Cells were collected, washed,and stained with antibodies for the detection of the chimeric proteinbinding by flow cytometry. As shown in FIG. 7A and FIG. 7B), and asexpected, the chimeric proteins did not significantly bind the parentalcell lines. However, the hCD172a (SIRPα)-Fc-CD40L bound the HeLa/hCD47engineered cell line in a concentration-dependent manner; based on thisdata an EC₅₀ value of 21.51 nM was calculated (FIG. 7A). Similarly, themCD172a (SIRPα)-Fc-CD40L bound the CHOK1/mCD40 engineered cell line in aconcentration-dependent manner; based on this data an EC₅₀ value of 20.2nM was calculated.

Example 4: Characterization of the Binding Affinity of theCD172a(SIRPα)-Fc-CD40L Chimeric Protein by Surface Plasmon Resonance(SPR)

The binding affinity of the different domains of the hCD172a(SIRPα)-Fc-CD40L chimeric protein was measured by surface plasmonresonance (SPR) using the BioRad ProteOn XPR 360 system. Specifically,the affinity of the chimeric protein for CD47, FcγR1A, and FcRn wasdetermined and compared to recombinant control proteins, and the resultsare shown in FIG. 8A, to FIG. 8C, respectively. Kinetic data collectedis summarized in the table shown in FIG. 8D.

As shown in FIG. 8A, the CD172a (SIRPα)-Fc-CD40L chimeric protein boundto CD47 and with high affinity. The ‘on-rate’ of CD172a (SIRPα)-Fc-OX40Lto human CD47 was rapid, however the ‘off-rate’ was much lower, in fact˜40-fold slower than the ‘off-rate’ of recombinant CD47-Fc, indicatingthat CD172a (SIRPα)-Fc-OX40L bound quickly and stably, with longon-target residence time. The KD of CD172a (SIRPα)-Fc-OX40L binding tohuman CD47 was calculated to be 3.59 nM. The affinity measurementsdemonstrated high-affinity binding to the chimeric protein, exceptagainst Fc receptors with effector function (FIG. 8B and FIG. 8C).Importantly, the off-rates of the chimeric protein were much slower thanthose of benchmark control proteins; the chimeric protein dissociationfrom CD47 was 2.78 fold longer than CD172a (SIRPα)-Fc.

Additionally, the binding affinity of murine CD172a (SIRPα)-Fc-CD40L tomCD40 was assessed by SPR. It was determined that the chimeric proteinbound to mCD40 tightly at a Kd of 0.756 nM, as shown in the in FIG. 8E.

Example 5: Functional Assays of the CD172a (SIRPα)-Fc-CD40L ChimericProtein

Functional assays, ELISA-based blocking assay and macrophage engulfmentassays, were performed to demonstrate functional activity of the humanCD172a (SIRPα)-Fc-CD40L chimeric protein. ELISA-based blocking assay wasperformed to demonstrate that hCD172a (SIRPα)-Fc-CD40L chimeric proteinbinding to cells over-expressing human CD47 (HeLa/hCD47 andJurkat/endogenous-CD47) can be disrupted by pre-incubating cells with ahuman CD47 blocking antibody. HeLa cells stably transfected with a humanCD47-expressing plasmid, were incubated with increasing concentrationsof the human CD172a (SIRPα)-Fc-CD40L chimeric protein, alone, or afterHeLa/hCD47 cells were pre-incubated with a human CD47 blocking antibody(FIG. 9A, middle panel). HeLa/hCD47 bound the hCD172a (SIRPα)-Fc-CD40Lchimeric protein in a concentration-dependent manner (FIG. 9A, middlepanel, top curve). The binding was blocked when HeLa/hCD47 cells werepre-treated with the CD47 blocking antibody (FIG. 9A, middle panel,bottom curve).

Jurkat cells, which expressed high levels of human CD47 endogenously,bound the hCD172a (SIRPα)-Fc-CD40L chimeric protein in aconcentration-dependent manner (FIG. 9A, right panel, top curve).Similarly, this binding was blocked when Jurkat cells were pre-treatedwith the same CD47 blocking antibody (FIG. 9A, right panel, bottomcurve).

Together, these data indicated that the binding of the CD172a (SIRPα)component of hCD172a (SIRPα)-Fc-CD40L chimeric protein was highlyspecific to both over-expressed and endogenous CD47, since binding ofthe hCD172a (SIRPα)-Fc-CD40L chimeric protein was impeded when CD47access is blocked.

In a macrophage engulfment assay, primary derived human macrophages wereincubated with CD47-expressing cells in the presence or absence of thehCD172a (SIRPα)-Fc-CD40L chimeric protein, in order to assesssuppression of the ‘don't eat me’ signal produced when macrophage boundCD172a (SIRPα) was freely able to interact with CD47. A schematicshowing the basic principles of the macrophage engulfment assay is shownin FIG. 9B. Primary human monocytes were isolated and differentiated invitro into macrophages. As the donor of CD47, the suspension cell lineJurkat was identified as expressing high levels of membrane bound CD47.Macrophages and Jurkat cells were incubated with different cell tracedyes, and co-cultured, in the presence or absence of the CD172a(SIRPα)-Fc-CD40L chimeric protein (FIG. 9B). In the absence of thechimeric protein, Jurkat-CD47 interacted with macrophage-CD172a (SIRPα),blocking phagocytosis, resulting in baseline levels of cells which weredouble positive for both cell trace dyes (FIG. 9C, left bar). When thechimeric protein was added to the Jurkat/macrophage co-culture,increased levels of double positive cells were detected, which increasedin a concentration-dependent manner (FIG. 9C, right three bars). Theresults of the macrophage engulfment assay indicated that the CD172a(SIRPα)-Fc-CD40L chimeric protein was able to promote macrophagephagocytosis of CD47⁺ cells.

Example 6: Characterization of the Murine CD172a (SIRPα)-Fc-CD40LChimeric protein

The murine CD172a (SIRPα)-Fc-CD40L chimeric protein was characterized byperforming a Western blot analysis against each individual domain of thechimeric protein, i.e., via anti-CD172a (SIRPα), anti-Fc, and anti-CD40Lantibodies. As shown in FIG. 10A, all three domains of the mCD172a(SIRPα)-Fc-CD40L chimeric protein were detected under non-reduced (Lane2), reduced (Lane 3) and reduced+PNGase treatments (Lane 4). Thereduced, glycosylated form of the chimeric protein migrated at theexpected molecular weight of approximately 110 kDa. The reduced,deglycosylated form was not detected by any of the antibodies, whichcould be due to its dependence on the protein being glycosylated.

ELISA assays were performed to demonstrate the binding affinity of thedifferent domains of the mCD172a (SIRPα)-Fc-CD40L chimeric protein tointeract with their predicted binding partners (i.e., CD47 or CD40).Specifically, the CD172a (SIRPα) domain of the mCD172a (SIRPα)-Fc-CD40Lchimeric protein was detected by capturing to a plate-bound recombinantCD47 protein and detecting via an HRP-conjugated anti-Fc antibody (FIG.10B, left panel, square symbols). Recombinant mCD172a (SIRPα)-mFc wasused to generate a standard curve (FIG. 10B, left panel, circlesymbols). The CD40L domain of the chimeric protein was detected bycapturing to a plate-bound recombinant murine CD40 protein and detectingvia a CD40L-specific antibody (FIG. 10B, right panel, square symbols).Recombinant mCD40L was used to generate a standard curve (FIG. 10B,right panel, circle symbols). As shown in FIG. 10B, the differentdomains of the mCD172a (SIRPα)-Fc-CD40L chimeric protein effectivelyinteracted with their respective binding partners and with highaffinity.

The in vivo anti-tumor activity of the mCD172a (SIRPα)-Fc-CD40L chimericprotein was analyzed using the CT26 mouse colorectal tumor model. In oneset of experiments, Balb/c mice were inoculated with CT26 tumor cells onday 0 and/or rechallenged with a second inoculation of CT26 tumor cellsat day 30. Following 4 days of tumor growth, when tumors reached adiameter of 4-5 mm, mice were treated with anti-CD47, anti-CD40, acombination of the two antibodies, or with mCD172a (SIRPα)-Fc-CD40Lchimeric protein.

The tumor growth for each treatment group was assessed as shown in FIG.11A. Specifically, the untreated mice developed tumors quickly.Treatment with the anti-CD47, anti-CD40, or the combination of those twoantibodies appeared to slightly delay the development of tumors. Incomparison, treating mice with the mCD172a (SIRPα)-Fc-CD40L chimericprotein significantly prevented and/or delayed the development oftumors. Importantly, the CD172a (SIRPα)-Fc-CD40L chimeric protein iseffectively able to kill tumor cells and/or reduce tumor growth whenrechallenged (which illustrates a cancer relapse). Thus, the CD172a(SIRPα)-Fc-CD40L chimeric protein appears to generate a memory responsewhich may be capable of preventing relapse.

The overall survival percentage of mice through forty-five days aftertumor inoculation was also assessed. All of the untreated mice diedwithin twenty-one days after tumor inoculation. Most of the mice treatedwith a single antibody died around day 30. The mice receiving acombination of anti-CD40 and anti-CD47 antibodies demonstrated onlyabout a 30% survival at day 50. Significantly, all the mice treated withthe mCD172a (SIRPα)-Fc-CD40L chimeric protein survived past fifty daysafter tumor inoculation as shown in FIG. 11B.

As shown in FIG. 11C, monotherapy with either anti-CD40 or anti-CD47 ledto moderate extensions in the tumor growth rates for most mice, with oneanimal completely rejected the tumor (anti-CD40 group, out of twelvetotal mice treated). When the two antibodies were administered incombination, there was a synergistic effect, with two out six mice inthe long-term follow-up group having complete tumor rejection. For micetreated with mCD172a (SIRPα)-Fc-CD40L, there was significantly superioractivity in both the 150 and 300 μg dose levels as compared to theantibody combinations, with a slightly improved rejection rate in the150 versus the 300 μg group. In comparison to the antibody combinationtreatment, there was an 80% complete rejection rate observed withtreatment using the mCD172a (SIRPα)-Fc-CD40L chimeric protein.

Immune phenotyping was also performed by analyzing splenocytes, lymphnode cells, and tumor infiltrating lymphocytes on day thirteen posttumor inoculation. As shown in FIG. 12A, FIG. 12C, and FIG. 12D, micetreated with the mCD172a (SIRPα)-Fc-CD40L chimeric protein exhibitedhigher percentages of total CD4+ T cells in the spleen, peripheral lymphnodes and tumor as compared to the control or the combination antibodytreatment group (anti-CD40 and anti-CD47). Within the spleen, thisincrease mostly comprised an increase in CD4+CD25− T cells, which wouldbe consistent with the notion that activation of non-regulatory T cellswas involved (FIG. 126). There was also an increase in CD8+ T cells inmice treated with mCD172a (SIRPα)-Fc-CD40L in the peripheral lymphnodes, but this increase was not observed in the spleen or tumor (FIG.12A, FIG. 12C, and FIG. 12D).

The ability of the mCD172a (SIRPα)-Fc-CD40L chimeric protein tostimulate the recognition of tumor antigens by CD8+ T cells was alsoanalyzed. Specifically, FIG. 12E shows tetramer staining analysis fordetermining the fraction of CD8+ T cells that recognized the AH1 tumorantigen natively expressed by CT26 tumors. Within the spleen and tumorinfiltrated lymphocytes (TIL), a higher proportion of CD8+ T cells wasfound to recognize the AH1 tumor antigen in mice treated with thechimeric protein as compared to the untreated mice.

One of the indicators of CD40 activation was the proportion of cellswhich upregulated IL-15 receptor alpha. Amongst the treatment groups,there was a significant increase in the mice treated with the 150 μgdose of the mCD172a (SIRPα)-Fc-CD40L chimeric protein, but not in theanti-CD40/CD47 treatment group (FIG. 12F).

Together, these data demonstrate that mCD172a (SIRPα)-Fc-CD40L waseffective at eliminating established tumors and generating a memoryimmune response capable of preventing tumor growth upon re-challenge.Moreover, mCD172a (SIRPα)-Fc-CD40L outperformed the benchmark antibodycombinations (anti-CD47+anti-CD40) when administered at the optimaldose. Notably, the 150 μg dose of mCD172a (SIRPα)-Fc-CD40L appeared tobe more efficacious and with a better defined immune signature than the300 μg dose.

The above data clearly demonstrate, inter alia, functional activity ofmCD172a (SIRPα)-Fc-CD40L in vivo, at least, in treating cancer.

Example 7: Characterization of the Human CD172a (SIRPα)-Fc-CD40LChimeric Protein In Vivo

The inability of the human CD172a (SIRPα)-Fc-CD40L chimeric protein tobind to red blood cells and, thereby, causing hemolysis was then tested.Here, hCD172a (SIRPα)-Fc-CD40L was contacted with cynomolgus macaque redblood cells (RBCs; FIG. 13 left top panel). No significant change in RBCcounts was detected in the two monkeys; thus, the chimeric protein didnot cause significant lysis of cynomolgus macaque RBCs in vivo. ThehCD172a (SIRPα)-Fc-CD40L chimeric protein likewise did not appear tosignificantly affect indicators hemolysis (See FIG. 13, remainingpanels). No evidence of RBC lysis or platelet depletion was observedfollowing treatment with hCD172a (SIRPα)-Fc-CD40L. Gross safetyassessments were made multiple times daily, and no additional safetysignals were observed. These data indicate that hCD172a (SIRPα)-Fc-CD40Ldoes not cause hemolysis of RBCs as an unwanted side effect.

Example 8: Characterization of Combination Treatments with a Pluralityof Chimeric Proteins

In this example, anti-tumor potency and/or synergistic effects weredetermined for treatments comprising combinations of chimeric proteins.Specifically, murine models of colorectal cancer (MC38 or CT26) or ofmelanoma (B16.F10) were used to assess the effects of these treatmentson tumor growth and overall survival.

Balb/c mice (n=6, 8, or 9) described in FIG. 14 were inoculated in thehind flank with 2.5×10⁵ MC38-ova tumor cells. On days 5 and 8, mice weretreated with the OX86 (anti-OX40) antibody; the RMP1-14 or 29F.1.A12(anti-PD-1) antibody; the RMT3-32 (anti-TIM3) antibody; the anti-TIM3and the anti-OX40 antibodies; the anti-TIM3, the anti-OX40, and theanti-PD-1 (RMP1-14) antibodies; the anti-OX40, the anti-TIM3, and the29F.1.A12/anti-PD-1 antibodies; or the TIM3-Fc-OX40L chimeric protein.When provided, 100 μg of each antibody was administered viaintraperitoneal injection. When provided, 300 μg of the TIM3-Fc-OX40Lchimeric protein was administered via intraperitoneal injection on day 5and again on day 7. Tumor area was calculated on the 19th day afterinoculation by taking perpendicular tumor diameter measurements usingelectronic calipers. Neither the mice treated with a single antibody northe mice treated with both the anti-TIM3 and the anti-OX40 antibodieshad a statistically-significant reduction in tumor size relative tocontrol treatments. However, when compared to control treatments, astatistically-significant (p<0.05) reduction in tumor size was observedin mice treated with either combination of three antibodies and in micetreated with two sequential doses of the TIM3-Fc-OX40L chimeric protein.

Balb/c mice (n=8 or 9) described in FIG. 15A and FIG. 15B wereinoculated in the hind flank with CT26 tumor cells. Mice in a firsttreatment group, on days 5 and 7, were treated with 150 μg of theTIM3-Fc-OX40L chimeric protein. Mice in a second treatment group weretreated on day 5 and 7 with 150 μg of the TIM3-Fc-OX40L chimeric proteinand then treated on day 7 and 9 with 150 μg of the CD172a(SIRPα)-Fc-CD40L chimeric protein. Mice in a third treatment group weretreated on day 5 and 7 with 150 μg of the TIM3-Fc-OX40L chimeric proteinand then treated on day 7 and 9 with 150 μg of the CSF1R-Fc-CD40Lchimeric protein. Mice in a fourth treatment group were treated on day 5and 7 with 150 μg of the CD172a (SIRPα)-Fc-CD40L chimeric protein andthen treated on day 7 and 9 with 150 μg of TIM3-Fc-OX40L chimericprotein. Mice in a fifth treatment group were treated on day 5 and 7with 150 μg of the CSF1R-Fc-CD40L chimeric protein and then treated onday 7 and 9 with 150 μg of the TIM3-Fc-OX40L chimeric protein. Tumorareas were calculated periodically. Of the mice in the first, second,third, and fifth treatment groups, only one mouse in each treatmentgroup rejected the tumor inoculation and two mice in the fourth grouprejected the tumor inoculation.

As shown in FIG. 15A, mice in each of the treatment groups showedreductions in tumor size (relative to control treatments) over thecourse of the test period, with the greatest reduction observed for miceof the fourth treatment group, which were first treated with CD172a(SIRPα)-Fc-CD40L chimeric protein and then treated with theTIM3-Fc-OX40L chimeric protein. The next greatest tumor reduction wasfor the same CD172a (SIRPα)-Fc-CD40L pairing TIM3-Fc-OX40L yet in areversed order. Thus, for this paring, treating with the TIM3-Fc-OX40Lchimeric protein second provided the greater reduction in tumor size.Surprisingly, an opposite pattern was observed for the other pairing ofchimeric proteins. Indeed, a greater reduction in tumor size resultedwhen the TIM3-Fc-OX40L chimeric protein is treated before theCSF1R-Fc-CD40L when compared to a treatment with the CSF1R-Fc-CD40Lchimeric protein first. Thus, for this paring, treating with theTIM3-Fc-OX40L chimeric protein first provides the greater reduction intumor size. Finally, for the treatment groups in which TIM3-Fc-OX40L wastreated first, a greater reduction in tumor size was observed when adifferent chimeric protein was provided second relative to the mice whoreceive TIM3-Fc-OX40L as both a first and a second treatment. Thus, forcertain chimeric proteins, there may be an advantage from administeringdifferent first and second chimeric proteins.

Similarly, as shown in FIG. 15B, mice in each of the treatment group hadimproved survival relative to the control treated mice; with survival atabout 30 days of between about 50% and 75%. Again, for the treatmentgroups in which TIM3-Fc-OX40L was treated first, improved survival wasobserved when a different chimeric protein was provided second relativeto the mice who receive TIM3-Fc-OX40L as both a first and a secondtreatment. Again, for certain chimeric proteins, there may be anadvantage from administering different first and second chimericproteins.

These data demonstrate that treatments including sequential treatmentstwo chimeric proteins (either identical or different chimeric proteins)provides enhanced anti-tumor efficacy and improved survival. Moreover,for certain chimeric proteins, efficacy and survival may be affected bythe order that two different chimeric proteins are administered.Furthermore, for certain chimeric proteins, efficacy and survival may beaffected when the second chimeric protein administered differs from thefirst-administered chimeric protein. These data support theunderstanding that a combined regimen involving the administration ofone or more chimeric proteins which induce an innate immune responsebefore, concurrently with, or subsequent to administration of one ormore chimeric proteins which induce an adaptive immune response mayprovide synergistic effects (e.g., synergistic anti-tumor effects).

Example 9: Characterization of the Contribution of an Fc Domain in aLinker to Functionality of Chimeric Proteins

In this example, the contribution of an Fc domain in a linker tofunctionality of chimeric proteins of the present invention was assayed.Here, α PD-1-Fc-OX40L was used as a model for Fc-containing chimericproteins. Thus, the data presented below is relevant to chimericproteins of the present invention.

In its native state, PD-1 exists as monomer whereas OX40Ls tend todimerize due to electrostatic interactions between the OX40L domains; Fcdomains associate with each other via disulfide bonds, e.g., via theircysteine residue(s). Together, several inter-molecular interactions maycontribute to the quaternary structure of PD-1-Fc-OX40L. There are, atleast, four potential configurations of PD-1-Fc-OX40L, with the chimericprotein existing as a monomer, a dimer, a trimer, or a hexamer. See,FIG. 16.

The existence of monomeric and dimeric configurations of the chimericprotein was tested by exposing chimeric proteins to reducing andnon-reducing conditions and then running the proteins on SDS-PAGE. Undernon-reducing conditions (Reduced: “−”), the chimeric protein migrated inSDS-PAGE at about 200 kDa. Here, Western blots were probed withantibodies directed against PD-1, Fc, or OX40L in, respectively, theleft, middle, and right blots shown in FIG. 17. Since, the predictedmonomeric molecular weight of the chimeric protein is 57.6 kDa, the 200kDa species was expected to be, at least a dimer. However, under reducedconditions (Reduced: “+”), which reduces disulfide bonds (e.g., betweenFc domains), the chimeric protein migrated in SDS-PAGE at about 100 kDa.Since the 100 kDa species was heavier than expected, it was predictedthat the extra mass was due to glycosylation. Finally, chimeric proteinswere treated with Peptide-N-Glycosidase F (PNGaseF “+”) and run onSDS-PAGE under reduced conditions. Under these conditions, the chimericprotein migrated at about 57.6 kDa. These data suggest that the chimericprotein is glycosylated and exists naturally, at least, as a dimer; withdimerization likely due to disulfide bonding between Fc domains e.g.,via their cysteine residue(s).

SDS-PAGE gel methods do not accurately predict the molecular weight forhighly charged and/or large molecular weight proteins. Thus, chimericproteins were next characterized using Size Exclusion Chromatography(SEC). Unlike SDS-PAGE, in which the negatively-charged SDS reducescharge-based interactions between peptides, SEC does not use detergentsor reducing agents. When the PD-1-Fc-OX40L chimeric protein was run onSEC, none of the peaks were around 200 kDa. This suggests, thatnatively, the chimeric protein does not exist as a dimer. Instead, apeak having a size greater than 670 kDa was detected. See, FIG. 18. Thisand the prior data suggests that the PD-1-Fc-OX40L chimeric proteinexists as a hexamer in its native state.

As shown above, when run on SDS-PAGE under non-reducing conditions orunder reducing conditions, SDS in the sample and/or running bufferconverts the hexameric PD-1-Fc-OX40L chimeric protein into a predominantdimer or monomer, respectively, in the absence and presence of areducing agent. See, FIG. 19 (left gel). When run on native PAGE, whichlacks SDS, and in the absence of a reducing agent, the chimeric proteinexists as a hexamer. However, when run on native PAGE and in thepresence of a reducing agent (which reduces disulfide bonds) thechimeric protein migrated heavier than expected; as shown in FIG. 19(right gel, lane 2), with the chimeric protein failed to substantiallymigrate out of the loading well. This data suggests that the chimericprotein oligomerized into a higher-order protein. Thus, in chimericproteins, disulfide bonding appears to be important for controllinghigher-order oligomerization.

To further confirm this, chimeric proteins lacking an Fc domain wereconstructed, e.g., “PD-1-No Fc-OX40L”. Such chimeric proteins will nothave the disulfide bonding which occurs between Fc domains in thechimeric proteins described previously. As shown in FIG. 20, whenchimeric proteins lacking Fc domains are run on native PAGE, none of theprotein substantially migrated out of its loading well; again,suggesting that the “No Fc” chimeric proteins have formed aconcatemer-like complex comprising numerous proteins. Thus, omission ofthe Fc domain in a chimeric protein leads to formation of proteinaggregates. These data indicate that disulfide bonding, e.g., between Fcdomains on different chimeric proteins, stabilizes the chimeric proteinsand ensures that they each exist as a hexamer and not as a higher-orderprotein/concatemer. In other words, the Fc domain surprisingly putsorder to chimeric protein complexes. Lanes 1 to 4 respectively include2.5 μg, of PD-1-No Fc-OX40L, 5 μg of PD-1-No Fc-OX40L, 2.5 μg of PD-1-NoFc-OX40L, and 5 μg of PD-1-No Fc-OX40L

Shown in FIG. 21 is a model summarizing the above data and showing how ahexamer and concatemers form from chimeric proteins of the presentinvention. The illustrative chimeric protein (PD-1-Fc-OX40L) naturallyforms into a hexamer (due to electrostatic interactions between theOX40L domains and dimerization by Fc domains). However, in the absenceof the controlling effects off disulfide bonding between Fc domains,under reduced conditions for the PD-1-Fc-OX40L protein and due to theabsence of Fc domains in the PD-1-No Fc-OX40L, these latter chimericproteins form concatemers.

Additionally, chimeric proteins were constructed in which the Fc domain(as described herein) was replaced with Ficolin (which lacks cysteineresidues necessary for disulfide bonding between chimeric proteins). Aswith the No Fc chimeric proteins and chimeric proteins comprising an Fcand run on native PAGE and in the presence of a reducing agent (both ofwhich formed aggregates that do not migrate into a gel) chimericproteins comprising Ficolin appear to also form higher-order latticeswhich did not migrate into a gel. These data reinforce the conclusionthat disulfide binding is important for proper folding and function ofchimeric proteins of the present invention.

Finally, chimeric proteins were prepared using coiled Fc domains(CCDFc). Very little purified protein was delivered under functionalevaluation.

Accordingly, including an Fc domain in a linker of a chimeric protein(which is capable of forming disulfide bonds between chimeric proteins),helps avoid formation of insoluble and, likely, non-functional proteinconcatemers and/or aggregates.

Example 10: Characterization of Different Joining Linker Sequences forthe Chimeric Proteins

Different unique joining linker sequences (17 linkers) were identifiedwith varying characteristics (length, solubility, charge andflexibility). Constructs were then synthesized incorporating each ofthose 17 joining linker sequences into the ‘linker 2’ position, wherethe configuration of chimeric protein:

-   -   ECD 1—Joining Linker 1—Fc—Joining Linker 2—ECD 2

The production levels for those 17 constructs were tested in CHO cells.The following table provides a summary for the different joining linkersequences, characteristics of those joining linkers, the productionlevel (by A280), and the binding values (EC₅₀) based on FACS analysis toPD-L1 or OX40. Some variations in production levels and activity betweencertain joining linker sequences were determined.

TABLE 2 Summary for optional joining linker sequences HeLa- ProteinCHO-PD-L1 OX40 Joining Linker 2 Protein Name conc. A280 EC50 (nM)EC50 (nM) Sequence Characteristics PD-1_IgG4_OX40L (1) 0.17 27 6IEGRMD (SEQ Linker ID NO: 51) PD-1_IgG4_OX40L (2) 0.12 23 67 SKYGPPCPPCPIgG4 Hinge (SEQ ID NO: 49) Region PD-1_IgG4_OX40L (3) 0.15 25 140GGGSGGGS Flexible (SEQ ID NO: 54) PD-1_IgG4_OX40L (4) 0.11 36 125GGGSGGGGSG Flexible GG (SEQ ID NO: 55) PD-1_IgG4_OX40L (5) 0.22 25 41EGKSSGSGSES KST (SEQ ID Flexible + soluble NO: 56) PD-1_IgG4_OX40L (6)0.12 26 171 GGSG (SEQ ID Flexible NO: 57) PD-1_IgG4_OX40L (7) 0.11 27195 GGSGGGSGGG Flexible SG (SEQ ID NO: 58) PD-1_IgG4_OX40L (8) 0.21 2048 EAAAKEAAAKE Rigid Alpha Helix AAAK (SEQ ID NO: 59)PD-1_IgG4_OX40L (9) 0.23 45 87 EAAAREAAARE Rigid Alpha Helix AAAREAAAR(SEQ ID NO: 60) PD-1_IgG4_OX40L (10) 0.13 52 62 GGGGSGGGGS FlexibleGGGGSAS (SEQ ID NO: 61) PD-1_IgG4_OX40L (11) 0.07 25 100 GGGVPRDCGFlexible (SEQ ID NO: 52) PD-1_IgG4_OX40L (12) 0.11 33 70 GGGGAGGGGFlexible (SEQ ID NO: 62) PD-1_IgG4_OX40L (13) 0.12 38 60 GS (SEQ ID NO:Highly flexible 63) PD-1_IgG4_OX40L (14) 0.18 25 70 GSGSGS (SEQHighly flexible ID NO: 64) PD-1_IgG4_OX40L (15)  0.19 24 67 GSGSGSGSGSHighly flexible (SEQ ID NO: 65) PD-1_IgG4_OX40L (16) 0.11 34 77GGGGSAS (SEQ Flexible ID NO: 66) PD-1_IgG4_OX40L (17) 0.19 32 44APAPAPAPAPA Rigid PAPAPAPAP (SEQ ID NO: 67)

Characterization of PD-1-IgG4-OX40L chimeric proteins with differentjoining linker sequences (17 linkers) by Western blot analysis is shownin FIG. 22A to FIG. 22Q. Specifically, each individual domain of thefusion construct was probed using an anti-PD-1, anti-Fc, or anti-OX40Lantibody. Results showed similar performance across each chimericprotein suggesting that all of the candidate joining linker sequenceswere functional.

Additionally, each purified protein with different linker sequences wasalso characterized by binding to PD-L1 or OX40 in ELISA assays (FIG.23), as well as cell-based flow cytometry assays (FIG. 24A to FIG. 24P).

Example 11: Characterization of Murine PD-1-Fc-OX40L Chimeric Proteins

Tumor cells may express PD-L1 on their cell surface, which can bind toPD-1 expressed by a T cell (FIG. 25A and FIG. 25B). This interactionsuppresses activation of T cells. A chimeric protein comprising theextracellular domain of PD-1, adjoined to the extracellular domain ofOX40L (i.e., PD-1-Fc-OX40L) may bind to PD-L1 on the surface of a tumorcell, preventing binding to PD-1 on the surface of a T cell (FIG. 25C).The chimeric protein may then “dangle” from the surface of the tumorcell, and the OX40L portion of the chimeric protein may then bind toOX40 expressed on the surface of the T cell. This would result inreplacement of an inhibitory PD-L1 signal with a co-stimulatory OX40Lsignal to enhance the anti-tumor activity of T cells.

The binding affinity of the different domains of the murinePD-1-Fc-OX40L chimeric protein was measured by surface plasmon resonance(SPR) using the BioRad ProteOn XPR 360 system. Specifically, theaffinity of the chimeric proteins for PD-L1, PD-L2, OX40, and FcRn weredetermined and compared to recombinant control proteins, and the resultsare shown in the Table below:

Ka Kd KD (on-rate; (off-rate; (binding; Binding to: Sample 1/Ms) 1/s) M)PD-L1 PD1-Fc 3.24E+4 1.08E−3 33.3 nM PD1-Fc-OX40L 4.97E+4 4.87E−4 9.8 nMPD-L2 PD1-Fc 4.79E+4 1.71E−3 23.1 nM PD1-Fc-OX40L 5.29E+4 5.61E−4 10.6nM OX40 OX40L-Fc 6.74E+5 7.12E−4 1.06 nM PD1-Fc-OX40L 3.19E+0 3.07E−89.62 nM FcRn IgG2A 3.00E+6 2.42E−2 8.08 nM PD1-Fc-OX40L 4.72E+4 2.62E−355.6 nM

mPD-1-Fc-OX40L bound to chip-bound mPD-L1-His (9.8 nM), PD-L2-His (10.6nM), OX40-His (9.62 nM), and FcRn-His (55.6 nM) using SPR. Binding ofcontrol proteins (PD-1-Fc, OX40L-Fc, and IgG2A) were also shown. Nobinding of mPD-1-Fc-OX40L was detected to FcγR1.

Additional analysis was carried out to determine whether themPD-1-Fc-OX40L chimeric protein could bind its targets on the surface ofliving cells. To assess mPD-1-Fc-OX40L binding to murine PD-L1, PD-L2,and OX40, the Chinese hamster ovary cell line, CHOK1, was transfected tostably express murine PD-L1, PD-L2, and OX40 (FIG. 26A). mPD-1-Fc-OX40Lchimeric protein was incubated with each parental and over-expressingcell line for 2 hours. Cells were collected, washed, and stained withantibodies for the detection of the chimeric protein binding by flowcytometry. All engineered cell lines (CHOK1/hPD-L1, CHOK1/hPD-L2, andCHOK1/hOX40) bound mPD-1-Fc-OX40L in a concentration-dependent manner atlow nM as shown in FIG. 26B. mPD-1-Fc-OX40L did not bind to parentalCHOK1 cells since they did not express detectable levels of human PD-L1,OX40, or PD-L2. However, nearly the entire population of CHO-K1-PD-L1,CHOK1-PD-L2, and Jurkat/hOX40 cells shifted significantly, indicatingthat the different components of the chimeric protein were capable ofbinding to its respective receptor/ligands on living cells (FIG. 26A andFIG. 26B).

The functional activity of mPD-1-Fc-OX40L chimeric protein was assessedusing the superantigen cytokine release assay. In this assay, increasingconcentrations of staphylococcus enterotoxin B (SEB) were used toactivate human peripheral blood leukocytes in the presence of varioustest agents. The quantity of TNFα or IL-2 secreted into the culturesupernatant was monitored as a functional readout in the ability of testagents to either block suppressive signaling events or co-stimulateimmune activating signals. As shown in FIG. 26C, the mPD-1-Fc-OX40Lchimeric protein induced secretion of IL2 at higher levels (top curve)in comparison of other test agents i.e., PD-1-Fc, OX40L-Fc, andPD-1-Fc/OX40L-Fc. However, as shown in FIG. 26D, the mPD-1-Fc-OX40Lchimeric protein and the PD-1-Fc/OX40L-Fc induced the highest level ofsecreted TNFα (top two curves) in comparison to other test agents:PD-1-Fc and OX40L-Fc. Media and IgG controls were used. Together, theseresults suggest that mPD-1-Fc-OX40L chimeric protein functionallyactivates primary leukocytes to release TNFα and IL2 in vitro.

FIG. 27A to FIG. 27F show results from in vivo tumor studiesdemonstrating that the mPD-1-Fc-OX40L chimeric protein has significantanti-tumor activity in a CT26 tumor rechallenge model. Mice inoculatedwith CT26 tumors were alternately treated with anti-PD-1, anti-PD-L1,anti-OX40, a combination of anti-PD-L1 and OX40 antibodies, acombination of anti-PD-1 and OX40 antibodies, with control antibodies,or with one of three doses of the mPD-1-Fc-OX40L chimeric protein (i.e.,100 μg, 150 μg and 300 μg) and on two occasions (see bottom panel).Specifically, Tumor inoculation occurred on day 0, first treatment onday 5, and second treatment on day 7; tumor re-challenge (implantationof a second tumor on the opposite flank without re-treatment with drug)occurred on day 30 in any mice that rejected the primary tumor. Micewere re-challenged with CT26 tumor cells. FIG. 27A shows the evolutionof tumor size over sixty-five days after tumor inoculation for eachgroup. Importantly, the PD-1-Fc-OX40L chimeric protein is effectivelyable to kill tumor cells and/or reduce tumor growth when rechallenged(which illustrates a cancer relapse). Thus, the PD-1-Fc-OX40L chimericprotein appears to generate a memory response which may be capable ofpreventing relapse.

FIG. 27B and FIG. 27C shows the overall survival percentage, andstatistics, of mice and tumor rejection through forty days after tumorinoculation. FIG. 27D shows changes in CD4+ T-cells, CD4+CD25− effectorT cells or CD4+CD25+ regulatory T cells in the tumor of mice treatedwith the chimeric protein and other benchmark antibodies. FIG. 27E showschanges in CD4+ T-cells, CD4+CD25− effector T cells or CD4+CD25+regulatory T cells in the spleen of mice treated with the chimericprotein and other benchmark antibodies. FIG. 27F summarizes treatmentoutcomes for each group. For FIG. 27D and FIG. 27E, cohorts of treatedmice were euthanized thirteen days after initial tumor inoculation.Tumors and spleens were isolated, dissociated, and analyzed forproportions of effector and non-effector/Treg populations by flowcytometry.

Overall, administration of mPD-1-Fc-OX40L significantly reduced tumorsize in the CT26 colorectal cancer model. Particularly, use ofmPD-1-Fc-OX40L resulted in greater tumor regression than the OX40agonist and PD-L1 blocking antibodies (FIG. 27G). mPD-1-Fc-OX40Loutperformed anti-OX40, anti-PD-1, or anti-PD-L1 antibodies and antibodycombinations at low dose (100 μg, FIG. 27G) and at a higher dose (300μg, FIG. 27H). Cytokine signature suggests that 100 μg was a sub-optimaldose. At a higher dose (300 μg chimeric protein vs. 2700 μg mAb),mPD-1-Fc-OX40L significantly outperformed anti-PD-1/L1+anti-OX40 mAbcombinations. In FIG. 27G and FIG. 27H, data identified as “ARC FusionProtein” refers to the mPD-1-Fc-OX40L chimeric protein.

The above data clearly demonstrate, inter alia, functional activity ofmPD-1-Fc-OX40L in vivo, at least, in treating cancer.

Example 12: Characterization of Human PD-1-Fc-OX40L Chimeric Proteins

ELISA (enzyme-linked immunosorbent assay) assays were developed todemonstrate the binding affinity of the different domains of the humanPD-1-Fc-OX40L chimeric protein (also referred to as SL-279252) to theirrespective binding partners. FIG. 28A shows the binding and detection ofhuman PD-1-Fc-OX40L chimeric protein to human IgG, the binding partnerfor Fc (square symbols). Human Ig (hIg) was used as a standard (circlesymbols). It was observed that in ELISA assays generally, using thecentral Fc region to detect chimeric proteins tended to underestimatethe actual protein content in a sample. Therefore, low level of thehPD-1-Fc-OX40L chimeric protein was detected compared to standard inthis assay. FIG. 28B shows the binding and detection of humanPD-1-Fc-OX40L chimeric protein to the receptor OX40, i.e., the bindingpartner for OX40L (square symbols). Recombinant OX40L-Fc was used togenerate a standard curve (circle symbols). FIG. 28C shows dual-bindingELISA assay demonstrating the ability of PD-1-Fc-OX40L to bind andengage both targets (PD-L1 and OX40-His) simultaneously. Increasingconcentrations of hPD-1-Fc-OX40L chimeric protein were incubated with afixed amount of plate-bound recombinant human PD-L1 protein. Thereafter,recombinant OX40-His protein or a control His-tagged protein (HVEM-His)was incubated with the complex and binding was detected via anHRP-conjugated anti-His antibody. The results clearly show thathPD-1-Fc-OX40L binds to PD-L1 and OX40 simultaneously and with highspecificity.

Additional analyses were carried out to determine whether hPD-1-Fc-OX40Lfusion protein could bind its targets on the surface of living cells invitro. To assess hPD-1-Fc-OX40L's binding to the human OX40 receptor,the human AML T cell line Jurkat was engineered to overexpress OX40,creating Jurkat/hOX40 cells (verified by flow cytometry; FIG. 29, rightpanel). To assess binding to PD-L1, the Chinese hamster ovary cell line,CHOK1, which does not express human PD-L1, was transfected to stablyexpress human PD-L1 (FIG. 29A, left panel). To assess binding to humanPD-L2, CHOK1 cells were transfected to stably express human PD-L2 (FIG.29A, middle panel). Human PD-1-Fc-OX40L chimeric protein was incubatedwith each parental cell line and each of the over-expressing cell linesfor two hours. Cells were collected, washed, and stained with antibodiesfor the detection of chimeric protein binding by flow cytometry. Forhistograms to the left and in the middle, parental CHOK1 (cellpopulation to the right) and CHOK1/hPD-L1 (cell population to the left)cells were assessed by flow cytometry using an hPD-L1 antibody or anhPD-L2 antibody, respectively. In the right histogram, parental Jurkatcells (cell population to the right) and Jurkat/hOX40 (cell populationto the left) were assessed by flow cytometry using a hOX40 antibody. Allengineered cell lines (CHOK1/hPD-L1, CHOK1/hPD-L2, and Jurkat/hOX40)bound hPD-1-Fc-OX40L in a concentration-dependent manner at low nM.

As shown in FIG. 29B to FIG. 29D, hPD-1-Fc-OX40L did not bind toparental CHO-K1 cells since they did not express detectable levels ofhuman PD-L1 or PD-L2. Similarly, hPD-1-Fc-OX40L did not bind to parentalJurkat cells since they did not express detectable levels of OX40.However, nearly the entire population of CHO-K1-PD-L1, CHOK1-PD-L2, andJurkat/hOX40 cells shifted significantly, indicating that the differentcomponents of the chimeric protein were each capable of binding itsrespective receptor/ligands on living cells. in vitro cell bindingaffinities of SL-279252-CHOK1/hPD-L1 at 26.11 nM, SL-279252-CHOK1/hPD-L2at 7.60 nM, and SL-279252-Jurkat/hOX40 at 6.28 nM.

Next, surface plasmon resonance (SPR) analysis was performed todetermine the affinity by which SL-279252 bound to hPD-L1, hPD-L2, andhOX40. Specifically, polyhistidine-tagged versions of recombinant humanPD-L1, PD-L2, or OX40 was bound to ProteOn HTG tris-NTA chips (BIORAD).SL-279252 was then flowed over the bound ligands over a time course anda relative index of ‘on-rate’ (Ka) and ‘off-rate’ (Kd) was generated tocalculate binding affinity (K_(D)) of SL-279252 to each partner.Recombinant human PD-1-Fc and OX40L-Fc were used as positive controlsfor binding. These controls have a relatively fast ‘on-rate’ and anequally fast ‘off-rate’, resulting in low nanomolar binding affinities.The results of SPR binding affinity demonstrated high-affinity bindingfor each portion of the fusion protein (except against Fc receptors witheffector function). Importantly, the off-rates of hPD-1-Fc-OX40L weremuch slower than those of benchmark control proteins: hPD-1-Fc-OX40Ldissociation from PD-L1 was 18 fold longer than PD-1-Fc, from PD-L2 was13.4 fold longer than PD-1-Fc, and from OX40 was 36.32 fold longerOX40L-Fc. Together, these results indicated that the hPD-1-Fc-OX40Lfusion protein had a long residence time when bound to PD-L1 or PD-L2.

The above data clearly demonstrates that the different domains of thehuman PD-1-Fc-OX40L fusion protein (SL-279252) bind their native bindingpartners (e.g., receptor or ligand; PD-L1, PD-L2, and OX40) on thesurface of a mammalian cell membrane.

To confirm that all three domains of the human PD-1-Fc-OX40L (SL-279252)are intact and recognizable by a protein detection assay, Western blotanalysis was performed on purified fusion protein which were probed withhuman anti-PD-1, anti-Fc, and anti-OX40L (FIG. 30). SL-279252 wasdetected by all three antibodies and when the protein was run underreducing conditions, migrated at approximately 75 kDa. Approximately 50%of the non-reduced protein ran as a dimer, which was a potentialadvantage, given the in vivo oligomerization associated with OX40Lsignaling and function. The predicted molecular weight for SL-279252 was60.3 kDa. The reduced fraction of SL-279252 was detected at a highermolecular weight, which, without wishing to be bound by theory, may bedue to glycosylation. This was verified by treating SL-279252 with aprotein deglycosylase, PNGase F. Following deglycosylation, the reducedfraction of SL-279252 migrated exactly at the predicted molecular weightof 60.3 kDa. This provided evidence that SL-279252 wasco/post-translationally modified through glycosylation, which playsessential roles in the proper folding and stability of proteins, andcell-to-cell adhesion (Dalziel M, Dwek RA. Science 2014; Maverakis E,Lebrilla CB. J Autoimmun. 2015).

Since the human PD-1-Fc-OX40L chimeric protein retained glycosylasemodifications, further analysis was performed to determine whether itsglycosylation status impacted its function. hPD-1-Fc-OX40L was treatedwith the deglycosylase PNGase F, and then its binding to hOX40 wasassessed in routine functional ELISA (FIG. 31A) and cell binding assayswith Jurkat cells expressing hOX40 (FIG. 31B). Jurkat cell lines that donot express hOX40 were used as control. Results indicated thatglycosylation did not play a significant role in hPD-1-Fc-OX40L bindingto its interacting partners.

Next, an ELISA-based blocking/competition assay was performed todemonstrate that hPD-1-Fc-OX40L could out-compete human PD-1-biotin forbinding to plate-bound recombinant human PD-L1. In this assay,recombinant human PD-L1 was coated on high-binding ELISA plates.Horseradish peroxidase (HRP) signal was produced using detection withrecombinant human PD-1-Biotin, followed by an avidin-HRP avidin. Asshown in FIG. 32A, in case of a negative control (a chimeric proteinthat does contain PD-L1 binding domain) the signal for PD-1-biotin wasnot disrupted (FIG. 32A, top curve/Square symbols). Notably,hPD-1-Fc-OX40L blocked PD-1-Biotin binding to PD-L1 (thereby decreasingHRP signal), in a concentration-dependent manner (FIG. 32A, bottomcurve/circle symbols). The results of this assay demonstrated thathPD-1-Fc-OX40L strongly competes with PD-1-biotin for binding torecombinant human PD-L1, with a calculated IC₅₀ of 6.68 nM.

A cytokine release, tumor co-culture assay was performed to demonstratethat the hPD-1-Fc-OX40L chimeric protein was capable of inducing theexpression of IL-2 in T cells. As shown in FIG. 32B, primary human CD3+T cells, in the presence or absence of hPD-1-Fc-OX40L, were incubatedwith PD-L1_(low) or PD-L1_(high) human tumor cells; thus, allowingassessment of the effector function and proliferation of T cells usingIL2 secretion and flow cytometry-based immune assessment. Specifically,human peripheral blood leukocytes were isolated by density gradientcentrifugation, followed by negative enrichment for CD3+ cells, andsubsequent activation with CD3/CD28 beads. The activated cells were thenco-cultured with either α PD-L1_(low) prostate cancer cell (human PC3)or α PD-L1_(high) lung adenocarcinoma cell (human HCC827) in thepresence of absence of hPD-1-Fc-OX40L (500 ng and 5 μg concentrations).The quantity of IL-2 produced and secreted into the cell culturesupernatant was then measured by ELISA (FIG. 32C). The hPD-1-Fc-OX40Linduced higher levels of secreted IL2 in PC3 cells (FIG. 32C, leftbundle) than in HCC827 cells (FIG. 32C, right bundle). As human T cellsproduced significantly more IL-2 when co-cultured with the PC3 cell linethan with the HCC827 cell line, this suggested that the quantity ofPD-L1 inhibited IL-2 production (FIG. 32C). When hPD-1-Fc-OX40L wasadded to the co-cultures, however, increased IL-2 production wasobserved in both co-culture systems. In addition to measuring the amountof IL-2 secreted, the activated T cells from the co-culture assay werecollected and analyzed by intracellular flow cytometry. These dataindicated that hPD-1-Fc-OX40L increased Ki67, IFNγ, and TNFα staining inboth CD4⁺ and CD8⁺ T cells (FIG. 32D).

Another functional assay to characterize the functional activity ofhPD-1-Fc-OX40L chimeric protein is the superantigen cytokine releaseassay. In this assay, increasing concentrations of staphylococcusenterotoxin B (SEB) were used to activate human peripheral bloodleukocytes in the presence of various test agents; a flow chart of thesteps is shown in FIG. 32E. The quantity of TNFα (FIG. 32F) or IL-2(FIG. 32G) secreted into the culture supernatant was monitored as afunctional readout of the ability of test agents to either blocksuppressive signaling events or co-stimulate immune activating signals.As shown in FIG. 32F and FIG. 32G, the hPD-1-Fc-OX40L chimeric proteininduced secretion of TNFα and the secretion of IL2 at higher levels (topcurves) in comparison to other test agents PD-1-Fc, OX40L-Fc, andPD-1-Fc/OX40L-Fc. Media and IgG controls were used. Together, theseresults suggest that hPD-1-Fc-OX40L chimeric protein functionallyactivated primary human leukocytes cells in vitro.

Finally, ELISA assays were performed to demonstrate the binding affinityand cross-reactivity of the human PD-1-Fc-OX40L chimeric protein to OX40from rhesus macaque and PD-L1 and PD-L2 from cynomolgus macaque. Asshown in FIG. 33A, human PD-1-Fc-OX40L chimeric protein specificallybound to plate-bound recombinant cmPD-L1 and the binding was detected byan HRP conjugated anti-hFc antibody. Human PD-1-Fc-OX40L chimericprotein was used from two different sources (square and trianglesymbols). Human CD120b-Fc-TGFb chimeric protein (inverted trianglesymbols) was used as a control while recombinant cmPD-1-hFc was used togenerate a standard curve (circle symbols). These results indicate thathPD-1-Fc-OX40L chimeric protein cross-reacted with cmPD-L1.

hPD-1-Fc-OX40L chimeric protein specifically bound to plate-boundrecombinant cmPD-L2 and the binding was detected via an HRP-conjugatedanti-Goat IgG binding to Goat anti-hOX40L antibody. Human PD-1-Fc-OX40Lchimeric protein was used from two different sources (FIG. 33B, squareand circle symbols). Human CD120b-Fc-TGFb chimeric protein (FIG. 33B,triangle symbols) was used as a control. As shown in FIG. 33C, thebinding of human PD-1-Fc-OX40L chimeric protein from two differentsources (square and circle symbols) and hCD120b-Fc-TGFb chimeric protein(triangle symbols) to a plate-bound rmOX40 was demonstrated. Binding wasdetected via an HRP-conjugated anti-Goat IgG binding to Goat anti-hOX40Lantibody. Accordingly, the above data demonstrates that hPD-1-Fc-OX40Lchimeric protein cross-reacted with cmPD-L1, cmPD-L2, and rmOX40.

Example 12: Characterization of the Murine TIM3-Fc-OX40L ChimericProtein

An in silico structure prediction of a monomeric TIM3-Fc-OX40L chimericprotein (SL-366252) having 548 amino acid residues was generated, with ap-value 5.1×10⁻¹⁷. The molecular weight of the monomeric protein waspredicted to be 61.6 kDa. A structure of the chimeric protein isprovided in FIG. 34.

Specifically, the structure prediction revealed that six positions (1%)may be disordered. Secondary structure prediction of the entire sequenceof the chimeric protein showed that the protein has the composition of3% α-helix (H), 43% β-sheet (E), and 51% coil (C). The GDT (globaldistance test) and uGDT (un-normalized GDT) for the absolute globalquality were also calculated for the chimeric protein to give an overalluGDT(GDT) of 481(87). The three-state prediction for solventaccessibility of the protein residues were 35% exposed (E), 49%intermediate (M), and 14% buried (B).

A murine TIM3-Fc-OX40L chimeric protein was constructed. The chimericprotein was characterized by performing a Western blot analysis againsteach individual domain of the chimeric protein, i.e., via anti-TIM3,anti-Fc, and anti-OX40L antibodies. The Western blots indicated thepresence of a dominant trimeric band in the non-reduced lanes (FIG. 35,lane 2 in each blot), which was reduced to a glycosylated monomeric bandin the presence of the reducing agent, β-mercaptoethanol (FIG. 35, lane3 in each blot). As shown in FIG. 35, lane 4 in each blot, the chimericprotein ran as a monomer at the predicted molecular weight of 61.6 kDain the presence of both a reducing agent (β-mercaptoethanol) and anendoglycosidase (PNGase).

Cell binding assays were performed to demonstrate the binding affinityof the different domains of the mTIM3-Fc-OX40L chimeric protein towardstheir respective binding partners on the surface of a mammalian cellmembrane. For the cell binding assays, immortalized cell lines wereengineered to stably express murine receptor OX40 (CHOK1-mOX40).Increasing concentrations of mTIM3-Fc-OX40L were incubated with eachparental (control) and over-expressing cell lines for 2 hours. Cellswere collected, washed, and stained with antibodies for the detection ofchimeric protein binding by flow cytometry. As shown in FIG. 36,mTIM3-Fc-OX40L bound to the engineered cell line (CHOK1-mOX40) in aconcentration-dependent manner with low nM affinity. Specifically, theCHOK1 parental cell line (bottom curve) was not responsive to increasingconcentrations of the mTIM3-Fc-OX40L chimeric protein as it did notoverexpress mOX40. In comparison, the CHOK1-mOX40 cell line, whichoverexpressed mOX40, bound to mTIM3-Fc-OX40L in aconcentration-dependent manner. The cell binding assay also indicatedthat mTIM3-Fc-OX40L bound to mOX40 with an affinity of 15.2 nM.

In vivo functional assays were performed to demonstrate the functionalactivity of the mTIM3-Fc-OX40L chimeric protein. Mice were inoculatedwith CT26 tumors on day 0. Once the tumors were palpable and at least4-6 mm in diameter, mice were treated with two doses of 150 μg of themTIM3-Fc-OX40L chimeric protein. Immunophenotyping was performed onvarious tissues collected from the mice on day 13 after implantation.

Immune profiling was performed on tumor-bearing mice treated with themurine TIM3-Fc-OX40L chimeric protein. As shown in FIG. 37A, micetreated with the mTIM3-Fc-OX40L chimeric protein exhibited higherpercentages of total CD4+ T cells in the spleen, peripheral lymph nodesand tumor (right bundle in FIG. 37A) as compared to the controltreatment groups (left bundle in FIG. 37A). Within the spleen and thetumor, this increase in CD4+ T cell population was mostly due to anincrease in CD4+CD25− effector T cells, consistent with the notion thatactivation of non-regulatory T cells was involved (FIG. 37B). Thetreated mice also exhibited a lower percentage of CD4+CD25+ regulatory Tcells, suggesting that regulatory T cells may be suppressed by thechimeric protein (FIG. 37B).

The ability of the chimeric protein to stimulate the recognition oftumor antigens by CD8+ T cells was also analyzed. Specifically, FIG. 37Cshows tetramer staining analysis for determining the fraction of CD8+ Tcells that recognized the AH1 tumor antigen natively expressed by CT26tumors. Within the spleen, a higher proportion of CD8+ T cells was foundto recognize the AH1 tumor antigen in mice treated with themTIM3-Fc-OX40L chimeric protein (right bundle in FIG. 37C) as comparedto the untreated mice (left bundle in FIG. 37C). Notably, a much higherproportion of the AH1 tetramer positive CD8+ T cells was observed withintumor infiltrated lymphocytes (TIL) for mice treated with the chimericprotein (right bundle in FIG. 37C) as compared to the untreated controlmice (right bundle in FIG. 37C).

The in vivo anti-tumor activity of the mTIM3-Fc-OX40L chimeric proteinwas analyzed using the MC38 and CT26 mouse colorectal tumor models. Inone set of experiments, Balb/c mice were inoculated with CT26 tumorcells on day 0 and/or rechallenged with a second inoculation of CT26tumor cells at day thirty. Following four days of tumor growth, whentumors reached a diameter of 4-5 mm, mice were treated with eithercontrol antibodies or 150 μg of the mTIM3-Fc-OX40L chimeric protein.Treatments were repeated on day seven. An analysis of the evolution oftumor size over forty-five days after tumor inoculation was conducted.

As shown in FIG. 38A, the untreated mice developed significant tumors,whereas none of the mice treated with the mTIM3-Fc-OX40L chimericprotein developed tumors of detectable size. Importantly, themTIM3-Fc-OX40L chimeric protein is effectively able to kill tumor cellsand/or reduce tumor growth when rechallenged (which illustrates a cancerrelapse). Thus, the mTIM3-Fc-OX40L chimeric protein appears to generatea memory response which may be capable of preventing relapse. Theoverall survival percentage of mice (FIG. 38B) through fifty days aftertumor inoculation shows that all of the untreated mice died withintwenty-one days after tumor inoculation, whereas mice treated themTIM3-Fc-OX40L chimeric protein showed a 100% survival rate at fiftydays after tumor inoculation. FIG. 38C summarizes the treatment outcomesfor each group.

The above data clearly demonstrate, inter alia, functional activity ofmTIM3-Fc-OX40L in vivo, at least, in treating cancer.

EQUIVALENTS

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

Those skilled in the art will recognize, or be able to ascertain, usingno more than routine experimentation, numerous equivalents to thespecific embodiments described specifically herein. Such equivalents areintended to be encompassed in the scope of the following claims.

INCORPORATION BY REFERENCE

All patents and publications referenced herein are hereby incorporatedby reference in their entireties.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.

As used herein, all headings are simply for organization and are notintended to limit the disclosure in any manner. The content of anyindividual section may be equally applicable to all sections.

1.-35. (canceled)
 36. A method of treating cancer, comprisingadministering to a subject in need thereof: (i) a first chimeric proteinof a general structure of:N terminus-(a)-(b)-(c)-C terminus, wherein: (a) is a first domaincomprising an extracellular domain of TIM3, (b) is a linker comprisingat least one cysteine residue capable of forming a disulfide bond, and(c) is a second domain comprising an extracellular domain of OX40L; and(ii) a second chimeric protein of a general structure of:N terminus-(a)-(b)-(c)-C terminus, wherein: (a) is a first domaincomprising an extracellular domain of CSF1R, (b) is a linker comprisingat least one cysteine residue capable of forming a disulfide bond, and(c) is a second domain comprising an extracellular domain of CD40L. 37.The method of claim 36, wherein the first chimeric protein isadministered before the second chimeric protein.
 38. The method of claim36, wherein the first chimeric protein is administered after the secondchimeric protein.
 39. The method of claim 36, wherein the linkercomprises a polypeptide selected from a flexible amino acid sequence, anIgG hinge region, or an antibody sequence.
 40. The method of claim 39,wherein the linker comprises hinge-CH2-CH3 Fc domain from IgG4.
 41. Themethod of claim 40, wherein the hinge-CH2-CH3 Fc domain is from humanIgG4.
 42. The method of claim 41, wherein the linker comprises an aminoacid sequence that is at least 95% identical to the amino acid sequenceof SEQ ID NO:
 45. 43. The method of claim 41, wherein the linkercomprises an amino acid sequence that is at least 95% identical to theamino acid sequence of SEQ ID NO:
 46. 44. The method of claim 41,wherein the linker comprises an amino acid sequence that is at least 95%identical to the amino acid sequence of SEQ ID NO:
 47. 45. The method ofclaim 41, wherein the linker further comprises at least one joininglinker selected from SEQ ID NOs: 48 to
 94. 46. The method of claim 45,wherein the linker comprises at least two joining linkers each joininglinker independently selected from SEQ ID NOs: 48 to 94; wherein onejoining linker is located N terminal to the hinge-CH2-CH3 Fc domain andanother joining linker is located C terminal to the hinge-CH2-CH3 Fcdomain.
 47. The method of claim 36, wherein: the extracellular domain ofTIM3 comprises an amino acid sequence that is at least 95% identical tothe amino acid sequence of SEQ ID NO: 2; the extracellular domain ofOX40L comprises an amino acid sequence that is at least 95% identical tothe amino acid sequence of SEQ ID NO: 4; the extracellular domain ofCSF1R comprises an amino acid sequence that is at least 95% identical tothe amino acid sequence of SEQ ID NO: 29; and/or the extracellulardomain of CD40L comprises an amino acid sequence that is at least 95%identical to the amino acid sequence of SEQ ID NO:
 12. 48. The method ofclaim 36, wherein: the first chimeric protein comprises an amino acidsequence that is at least 95% identical to the amino acid sequence ofSEQ ID NO: 5, and/or the second chimeric protein comprises an amino acidsequence that is at least 95% identical to the amino acid sequence ofSEQ ID NO:
 30. 49. A method of treating colon cancer, comprisingadministering to a subject in need thereof: (i) a first chimeric proteinof a general structure of:N terminus-(a)-(b)-(c)-C terminus, wherein: (a) is a first domaincomprising an extracellular domain of TIM3, (b) is a linker comprisingat least one cysteine residue capable of forming a disulfide bond, and(c) is a second domain comprising an extracellular domain of OX40L; and(ii) a second chimeric protein of a general structure of:N terminus-(a)-(b)-(c)-C terminus, wherein: (a) is a first domaincomprising an extracellular domain of CSF1R, (b) is a linker comprisingat least one cysteine residue capable of forming a disulfide bond, and(c) is a second domain comprising an extracellular domain of CD40L. 50.The method of claim 49, wherein the first chimeric protein isadministered before the second chimeric protein, or the first chimericprotein is administered after the second chimeric protein.
 51. Themethod of claim 49, wherein: the extracellular domain of TIM3 comprisesan amino acid sequence that is at least 95% identical to the amino acidsequence of SEQ ID NO: 2; the extracellular domain of OX40L comprises anamino acid sequence that is at least 95% identical to the amino acidsequence of SEQ ID NO: 4; the extracellular domain of CSF1R comprises anamino acid sequence that is at least 95% identical to the amino acidsequence of SEQ ID NO: 29; and/or the extracellular domain of CD40Lcomprises an amino acid sequence that is at least 95% identical to theamino acid sequence of SEQ ID NO:
 12. 52. The method of claim 49,wherein the linker comprises an amino acid sequence that is at least 95%identical to the amino acid sequence of SEQ ID NO: 45, SEQ ID NO: 46 orSEQ ID NO:
 47. 53. The method of claim 52, wherein the linker furthercomprises at least one joining linker selected from SEQ ID NOs: 48 to94.
 54. The method of claim 43, wherein the linker comprises at leasttwo joining linkers each joining linker independently selected from SEQID NOs: 48 to 94; wherein one joining linker is located N terminal tothe hinge-CH2-CH3 Fc domain and another joining linker is located Cterminal to the hinge-CH2-CH3 Fc domain.
 55. The method of claim 53,wherein: the first chimeric protein comprises an amino acid sequencethat is at least 95% identical to the amino acid sequence of SEQ ID NO:13 or SEQ ID NO: 5, and/or the second chimeric protein comprises anamino acid sequence that is at least 95% identical to the amino acidsequence of SEQ ID NO: 30.